US20040077536A1

US20040077536A1 - Human and rat pgc-3, ppar-gamma coactivations and splice variants thereof

Info

Publication number: US20040077536A1
Application number: US10/380,492
Authority: US
Inventors: Kevin Hart; Carl Montague; Antonio Vidal-Puig
Original assignee: AstraZeneca AB
Current assignee: AstraZeneca AB
Priority date: 2000-09-15
Filing date: 2001-09-12
Publication date: 2004-04-22
Also published as: WO2002022818A1; AU2001287855A1; EP1320601A1; US7306922B2; US20090142351A1; JP2004508826A; GB0022670D0

Abstract

A gene PGC-3 PPAR gamma coactivator-3, and it's role in regulating the transcriptional activity of peroxisome proliferator activated receptor-Y PPAR-Y in adipose tissue. PGC-3 is highly expressed in human white adipose tissue and has utility in the development of new therapeutic agents for use in the treatement of obesity and other related disorders such as non-insulin dependent diabetes mellitus, insulin resistance syndrome, dyslipidemia, and atherosclerosis.

Description

This invention relates to the regulation of metabolism and in particular to human genes involved in obesity. The invention further relates to proteins encoded by the genes and to means of regulating their biological activity. In addition the invention relates to the use of the genes and proteins to identify therapeutic agents for controlling obesity and other related disorders such as non-insulin dependent diabetes mellitus (NIDDM), insulin resistance syndrome, dyslipidemia, and atherosclerosis.

Obesity results from an excessive accumulation of adipose tissue and is a growing public health problem in developed and developing countries. Highly overweight individuals show significant increases in the occurrence of NIDDM, coronary heart disease, some cancers and digestive diseases.

Current treatment is unsatisfactory and new drugs need to be developed. A major problem is that the mechanisms regulating obesity, and the role of increased adiposity in the development of metabolic dysfunction are unclear. What is apparent is that obesity results from an imbalance between energy intake and expenditure. Energy expenditure can be affected by alterations in basal metabolism, physical activity and adaptive thermogenesis.

Peroxisome proliferator-activated receptor-γ (PPARγ) is a recently identified member of the peroxisome proliferator-activated receptor family of nuclear hormone receptors (Tontonoz et al., Genes Dev. (1994) 8, 1224-1234). The expression of this protein is induced very early in the adipocyte differentiation process and, when expressed ectopically in fibroblastic cells, induces adipogenesis in response to activators of the receptor. Synthetic and naturally occurring ligands for PPARγ have been identified. Thiazolidinediones CIZDs), a class of insulin sensitising agents which are used for the treatment of NIDDM, have been shown to bind to and activate PPARγ. TZDs promote adipocyte differentiation of murine and human preadipocytes to mature, fat storing adipocytes. TZD activation of PPARγ has also been shown to regulate transcription of many adipocyte genes. In addition to the presence of a ligand, the activity of PPARγ has been shown to be influenced by the presence of coactivators and corepressors. When co-expressed in cells alongside PPARγ these proteins have been shown to greatly increase or repress the transcriptional activity of PPARγ. Differences in expression of these coactivators and corepressors between cell types may explain the observed differences in PPARγ mediated transcriptional activity between cells from different tissues.

One such coactivator is PGC-1 (Puigserver et al., Cell (1998) 92, 829-839). The expression of this 90 kDa nuclear protein is greatly increased in muscle and brown fat of mice upon their exposure to cold temperatures. Co-expression of PGC-1 with PPARγ has been shown to activate aspects of the adaptive thermogenic program.

However, PGC-1 is not expressed in white adipose tissue which makes up the majority of adipose tissue found in humans. The identification of a protein which regulates the activity of PPARγ in white adipose tissue is thus of great importance in understanding the development of human obesity.

In the present invention we disclose the cloning and identification of PGC-3, and its role in regulating the transcriptional activity of PPARγ in adipose tissue. PGC-3 is highly expressed in human white adipose tissue and shares sequence homology with PGC-1 in domains known to be responsible for distinct activity of the protein. Two distinct variants of PGC-3 have been identified, termed PGC-3a and PGC-3b, which arise from alternative splicing of the PGC-3 gene. A further splice variant has been identified, termed PGC-3c. Full length cDNA and protein sequences for each of the splice variants are provided. The invention further discloses that PGC-3 has utility in the development of new therapeutic agents for use in the treatment of obesity and other related disorders such as non-insulin dependent diabetes mellitus, insulin resistance syndrome, dyslipidemia, and atherosclerosis. The invention further provides methods for the identification of such therapeutic agents.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. All publications and patents referred to herein are incorporated by reference.

The term “PGC-3” as used herein encompasses both of the splice variants, PGC-3a and PGC-3b, as well as PGC-3c.

According to one aspect of the present invention we provide an isolated and purified polynucleotide molecule comprising a nucleic acid sequence which encodes a polypeptide having at least about 90% homology to a member selected from any one of

(a) (SEQ ID NO:2, SEQ ID NO:2 positions 1-600, SEQ ID NO:2 positions 400-1002, and SEQ ID NO:2 positions 200-800)

or

(b) (SEQ ID NO:4, SEQ ID NO:4 positions 1-600, SEQ ID NO:4 positions 400-996, and SEQ ID NO:4 positions 200-800)

or

(c) (SEQ ID NO:8, SEQ ID NO:8 positions 1-600, SEQ ID NO:4 positions 400-1023, and SEQ ID NO:4 positions 200-800

Isolated and purified polynucleotides of the present invention include sequences which comprise the human PGC-3a cDNA sequence set out in SEQ ID NO:1 and the human PGC-3b cDNA sequence set out in SEQ ID NO:3 and the human PGC-3c cDNA sequence set out in SED ID NO:7.

In addition we have also identified and sequenced a rat clone having a high degree of homology to PGC-3 (cf. Example 5). Polynucleotide and polypeptide molecules based on the rat PGC-3 sequence may be used by analogy with the human sequences. The rat sequence shows a high degree of sequence homology (78% sequence identity and rats are therefore expected to be useful in animal models of metabolism.

Therefore in a further aspect of the invention we provide an isolated and purified polynucleotide molecule comprising a nucleic acid sequence which encodes a polypeptide having at least about 90% homology to any one of SEQ ID NO:9, SEQ ID NO:9 positions 1-600, SEQ ID NO:2 positions 400-990, and SEQ ID NO:9 positions 200-800.

In a further aspect of the invention we provide fragments of the isolated and purified polynucleotide molecules of the present invention. By fragments we mean contiguous regions of the polynucleotide molecule including complementary DNA and RNA sequences, starting with short sequences useful as probes or primers of say about 8-50 bases, such as 10-30 bases or 15-35 bases, to longer sequences of up to 50, 100, 200, 500 or 1000 bases. Indeed any convenient fragment of the polynucleotide molecule may be a useful fragment for further research, therapeutic or diagnostic purposes. Further convenient fragments include those whose terminii are defined by restriction sites within the molecule of one or more kinds, such as any combination of Rsa1, Alu1 and Hinf1.

In a further aspect we provide homologues and orthologues of the isolated and purified polynucleotide molecules of the present invention. Preferred homologues and orthologues are polynucleotide molecules which display greater than 80% sequence homology, conveniently greater than 85%, for example 90%, to the PGC-3 cDNA sequences set out in SEQ ID NO:1 and SEQ ID NO:3. A homologue may be a polynucleotide molecule from the same species i.e. a homologous family member, alternatively, the homologue may be a similar polynucleotide molecule from a different species such as human, useful in developing new therapies for the treatment of IRS and other related disorders such as NIDDM, obesity and atherosclerosis. By the term orthologue we mean a functionally equivalent molecule in another species. The full sequences of the individual homologues and orthologues may be determined using conventional techniques such as hybridisation, PCR and sequencing techniques, starting with any convenient part of the sequence set out in SEQ ID NO: 1 or SEQ ID NO:3.

In a further aspect of the invention we provide isolated and purified polynucleotide molecules capable of specifically hybridising to the polynucleotide molecules of the present invention. By specifically hybridising we mean that the polynucleotide hybridises by base-pair interactions, under stringent conditions, to the polynucleotide molecules of the present invention or to the corresponding complementary sequences. Experimental procedures for hybridisation under stringent conditions are well known to persons skilled in the art. For example, hybridisation filters may be incubated overnight at 42° C. in a solution comprising 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6) 5×Denhardt's solution, 10% dextran sulphate, and 20 μg/ml denatured salmon sperm DNA; followed by washing the filters in 0.1×SSC at about 65° C. Hybridisation techniques are thoroughly described in Sambrook J., Fritsch E. F. and Maniatis T., Molecular Cloning a Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989.

In a further aspect we provide an expression vector comprising a polynucleotide molecule of the present invention.

A variety of mammalian expression vectors may be used to express the recombinant polypeptides of the present invention. Commercially available mammalian expression vectors which are suitable for recombinant expression include, pcDNA3 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593), pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), IZD35 (ATCC 37565), pLXIN, pSIR (CLONTECH), and pIRES-EGFP (CLONTECH).

Baculoviral expression systems may also be used with the present invention to produce high yields of biologically active polypeptides. Preferred vectors include the CLONTECH, BacPak™ Baculovirus expression system and protocols which are commercially available (CLONTECH, Palo Alto, Calif.).

Further preferred vectors include vectors for use with the mouse erythroleukaemia cell (MEL cell) expression system comprising the human beta globin gene locus control region (Davies et al., J. of Pharmacol. and Toxicol. Methods 33, 153-158).

Vectors comprising one or more polynucleotide molecules of the present invention may then be purified and introduced into appropriate host cells. Therefore in a further aspect we provide a transformed host cell comprising a polynucleotide molecule of the present invention.

The polypeptides of the present invention may be expressed in a variety of hosts such as bacteria, plant cells, insect cells, fungal cells and human and animal cells. Eukaryotic recombinant host cells are especially preferred. Examples include yeast, mammalian cells including cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including Drosophila and silkworm derived cell lines. Cell lines derived from mammalian species which may be used and which are commercially available include, L cells L-M(TK-) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), HEK 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).

The expression vector may be introduced into host cells to express a polypeptide of the present invention via any one of a number of techniques including calcium phosphate transformation, DEAE-dextran transformation, cationic lipid mediated lipofection, electroporation or infection

The transformed host cells are propagated and cloned, for example by limiting dilution, and analysed to determine the expression level of recombinant polypeptide. Identification of transformed host cells which express a polypeptide of the present invention may be achieved by several means including immunological reactivity with antibodies described herein and/or the detection of biological activity.

Polypeptides of the present invention may be expressed as fusion proteins, for example with one or more additional polypeptide domains added to facilitate protein purification. Examples of such additional polypeptides include metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilised metals (Porath, J., Protein Exp. Purif. 3:263 (1992)), protein A domains that allow purification on immobilised immunoglobulin, and the domain utilised in the FLAGS extension/affinity purification system (Immunex Corp, Seattle Wash.). The inclusion of cleavable linker sequences such as Factor XA or enterokinase (Invitrogen, San Diego Calif.) between the purification domain and the coding region is useful to facilitate purification. A preferred protein purification system is the CLONTECH, TALON™ nondenaturing protein purification kit for purifying 6xHis-tagged proteins under native conditions (CLONTECH, Palo Alto, Calif.).

Therefore in a further aspect we provide a method for producing a polypeptide of the present invention, which method comprises culturing a transformed host cell comprising a polynucleotide of the present invention under conditions suitable for the expression of said polypeptide.

In a further aspect of the present invention we provide a purified polypeptide comprising the human PGC-3a amino acid sequence set out in SEQ ID NO.2 or a variant of SEQ ID NO.2 having at least about 90% homology to a member selected from (SEQ ID NO.2 positions 1-600, SEQ ID NO.2 positions 400-1002, SEQ ID NO.2 positions 200-800), or a biologically active fragment thereof.

In a further aspect of the present invention we provide a purified polypeptide comprising the human PGC-3b amino acid sequence set out in SEQ ID NO.4 or a variant of SEQ ID NO.4 having at least about 90% homology to a member selected from (SEQ ID NO.4 positions 1-600, SEQ ID NO.4 positions 400-996, SEQ ID NO.4 positions 200-800), or a biologically active fragment thereof.

In a further aspect of the present invention we provide a purified polypeptide comprising the human PGC-3c amino acid sequence set out in SEQ ID NO.8 or a variant of SEQ ID NO.8 having at least about 90% homology to a member selected from (SEQ ID NO.8 positions 1-600, SEQ ID NO.8 positions 400-1023, SEQ ID NO.8 positions 200-800), or a biologically active fragment thereof.

In a further aspect of the present invention we provide a purified polypeptide comprising the rat PGC-3 amino acid sequence set out in SEQ ID NO.10 or a variant of SEQ ID NO.10 having at least about 90% homology to a member selected from (SEQ ID NO.10 positions 1-600, SEQ ID NO.10 positions 400-990, SEQ ID NO.10 positions 200-800), or a biologically active fragment thereof.

A variant is a polynucleotide or polypeptide which differs from a reference polynucleotide or polypeptide, but which retains some of its essential characteristics. For example, a variant of a PGC-3 polypeptide may have an amino acid sequence that is different by one or more amino acid substitutions, deletions and/or additions. The variant may have conservative changes (amino acid similarity), wherein a substituted amino acid has similar structural or chemical properties, for example, the replacement of leucine with isoleucine. Alternatively, a variant may have nonconservative changes, e.g., replacement of a glycine with a tryptophan. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted and the effect this will have on biological activity may be reasonably inferred from the present disclosure by a person skilled in the art and may further be found using computer programs well known in the art, for example, DNAStar software.

Amino acid substitutions may be made, for instance, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues. Negatively charged amino acids, for example, include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine; asparagine, glutamine; serine, threonine, phenylalanine, and tyrosine.

Suitable substitutions of amino acids include the use of a chemically derivatised residue in place of a non-derivatised residue. D-isomers and other known derivatives may also be substituted for the naturally occurring amino acids. See, e.g., U.S. Pat. No. 5,652,369, Amino Acid Derivatives, issued Jul. 29, 1997. Example substitutions are set forth in Table 1.

“Homology” as used in this description is a measure of the similarity or identity of nucleotide sequences or amino acid sequences. In order to characterise the homology, subject sequences are aligned so that the highest order identity match is obtained. Identity can be calculated using published techniques. Computer program methods to determine identity between two sequences, for example, include DNAStar software (DNAStar Inc., Madison, Wis.); the GCG program package (Devereux, J., et al., Nucleic Acids Research 1984, 12(1):387); and BLASTP, BLASTN, FASTA (Atschul, S. F. et al., J Molec Biol 1990, 215:403). Homology as defined herein is determined conventionally using the well known computer program, BESTFIT (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis., 53711). When using BESTFIT or another sequence alignment program to determine the similarity of a particular sequence to a reference sequence, the parameters are typically set such that the percentage identity is calculated over the full length of the reference nucleotide sequence or amino acid sequence and that gaps in homology of up to about 10% of the total number of nucleotides or amino acid residues in the reference sequence are allowed.

In a further aspect we provide polymorphic variants of the polynucleotides and polypeptides of the present invention. Polymorphisms are variations in polynucleotide or polypeptide sequences between one individual and another. DNA polymorphisms may lead to variations in amino acid sequence and consequently to altered protein structure and functional activity. Polymorphisms may also affect mRNA synthesis, maturation, transport and stability. Polymorphisms which do not result in amino acid changes (silent polymorphisms) or which do not alter any known consensus sequences may nevertheless have a biological effect, for example by altering mRNA folding or stability.

Knowledge of polymorphisms may be used to help identify patients most suited to therapy with particular pharmaceutical agents (this is often termed “pharmacogenetics”). Pharmacogenetics may also be used in pharmaceutical research to assist the drug selection process. Polymorphisms may be used in mapping the human genome and to elucidate the genetic component of diseases. The reader is directed to the following references for background details on pharmacogenetics and other uses of polymorphism detection: Linder et al. (1997), Clinical Chemistry, 43, 254; Marshall (1997), Nature Biotechnology, 15, 1249; International Patent Application WO 97/40462, Spectra Biomedical; and Schafer et al. (1998), Nature Biotechnology, 16, 33.

The polypeptides of the present invention may be genetically engineered in such a way that their interaction with other intracellular and membrane associated proteins are maintained but their effector function and biological activity are removed. A polypeptide genetically modified in this way is known as a dominant negative mutant. In the construction of a dominant negative mutant at least one amino acid residue position at a site required for activity in the native peptide is changed to produce a peptide which has reduced activity or which is devoid of detectable activity. Overexpression of the dominant negative mutant in an appropriate cell type down-regulates the effect of the endogenous polypeptide, thereby revealing the biological mechanisms involved in the control of metabolism.

Similarly, the polypeptides of the present invention may be genetically engineered in such a way that their effector function and biological activity are enhanced. The resultant overactive polypeptide is known as a dominant positive mutant. At least one amino acid residue position at a site required for activity in the native peptide is changed to produce a peptide which has enhanced activity. Overexpression of a dominant positive mutant in an appropriate cell type amplifies the response of the endogenous native polypeptide highlighting the regulatory mechanisms controlling cell metabolism.

Therefore in a further aspect we provide dominant negative and dominant positive mutants of the polypeptides of the present invention.

Novel sequences disclosed herein, may be used in another embodiment of the invention to regulate expression of PGC-3 genes in cells by the use of antisense constructs. For example an antisense expression construct may be readily constructed using the pREP10 vector (Invitrogen Corporation). Transcripts are expected to modulate translation of the gene in cells transfected with the construct. Antisense transcripts are effective for modulating translation of the native gene transcript, and are capable of altering the effects (e.g., regulation of tissue physiology) herein described. Oligonucleotides which are complementary to and hybridisable with any portion of mRNA disclosed herein are contemplated for therapeutic use. U.S. Pat. No. 5,639,595, “Identification of Novel Drugs and Reagents”, issued Jun. 17, 1997, wherein methods of identifying oligonucleotide sequences that display in vivo activity are thoroughly described, is herein incorporated by reference. Antisense molecules may also be synthesised for use in antisense therapy, using techniques known to persons skilled in the art. These antisense molecules may be DNA, stable derivatives of DNA such as phosphorothioates or methylphosphonates, RNA, stable derivatives of RNA such as 2′-O-alkylRNA, or other oligonucleotide mimetics. U.S. Pat. No. 5,652,355, “Hybrid Oligonucleotide Phosphorothioates”, issued Jul. 29, 1997, and U.S. Pat. No. 5,652,356, “Inverted Chimeric and Hybrid Oligonucleotides”, issued Jul. 29, 1997, which describe the synthesis and effect of physiologically-stable antisense molecules, are incorporated by reference. Antisense molecules may be introduced into cells by microinjection, liposome encapsulation or by expression from vectors harboring the antisense sequence.

In a further aspect we provide an antibody specific for a polypeptide of the present invention.

Antibodies can be prepared using any suitable method, for example, purified polypeptide may be utilised to prepare specific antibodies. The term “antibodies” includes polyclonal antibodies, monoclonal antibodies, and the various types of antibody constructs such as for example F(ab′) ₂, Fab and single chain Fv. Antibodies are defined to be specifically binding if they bind the antigen with a K_aof greater than or equal to about 10⁷M⁻¹. Affinity of binding can be determined using conventional techniques, for example those described by Scatchard et al., Ann. N.Y. Acad Sci., 51:660 (1949).

Polyclonal antibodies can be readily generated from a variety of sources, for example, horses, cows, goats, sheep, dogs, chickens, rabbits, mice or rats, using procedures that are well-known in the art. In general, antigen is administered to the host animal typically through parenteral injection. The immunogenicity of antigen may be enhanced through the use of an adjuvant, for example, Freund's complete or incomplete adjuvant. Following booster immunisations, small samples of serum are collected and tested for reactivity to antigen. Examples of various assays useful for such determination include those described in: Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; as well as procedures such as countercurrent immuno-electrophoresis (CIEP), radioimmunoassay, radioimmunoprecipitation, enzyme-linked immuno-sorbent assays (ELISA), dot blot assays, and sandwich assays, see U.S. Pat. Nos. 4,376,110 and 4,486,530.

Monoclonal antibodies may be readily prepared using well-known procedures, see for example, the procedures described in U.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439 and 4,411,993; Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), (1980).

The monoclonal antibodies of the invention can be produced using alternative techniques, such as those described by Alting-Mees et al., “Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas”, Strategies in Molecular Biology 3: 1-9 (1990) which is incorporated herein by reference. Similarly, binding partners can be constructed using recombinant DNA techniques to incorporate the variable regions of a gene that encodes a specific binding antibody. Such a technique is described in Larrick et al., Biotechnology, 7: 394 (1989).

Once isolated and purified, the antibodies may be used to detect the presence of antigen in a sample using established assay protocols.

In a further aspect of the invention we provide a method for identifying a therapeutic agent capable of modulating the activity of PGC-3 for use in the regulation of metabolism, which method comprises:

(i) contacting a candidate compound modulator with a PGC-3 polypeptide comprising any one of

(a) the amino acid sequence set out in SEQ ID NO.2 or a variant of SEQ ID NO.2 having at least about 90% homology to a member selected from (SEQ ID NO.2 positions 1-600, SEQ ID NO.2 positions 400-1002, SEQ ID NO.2 positions 200-800) or a biologically active fragment thereof;

or

(b) the amino acid sequence set out in SEQ ID NO.4 or a variant of SEQ ID NO.4 having at least about 90% homology to a member selected from (SEQ ID NO.4 positions 1-600, SEQ ID NO.4 positions 400-996, SEQ ID NO.4 positions 200-800) or a biologically active fragment thereof;

or

(c) the amino acid sequence set out in SEQ ID NO.8 or a variant of SEQ ID NO.8 having at least about 90% homology to a member selected from (SEQ ID NO.8 positions 1-600, SEQ ID NO.8 positions 400-996, SEQ ID NO.8 positions 200-800) or a biologically active fragment thereof;

and

(ii) measuring an effect of the candidate compound modulator on the activity of the PGC-3 polypeptide.

Activity as used herein refers to the ability of the therapeutic agent to mediate cell processes related to insulin resistance syndrome and other related disorders such as non-insulin dependent diabetes mellitus, dyslipidemia, obesity and atherosclerosis.

Modulation of the activity of PGC-3 comprises either stimulation or inhibition. Thus a therapeutic agent capable of modulating the activity of PGC-3 is an agent that either stimulates or inhibits the activity of PGC-3. The terms “modulator of PGC-3 activity” and “PGC-3 modulator” are also used herein to refer to an agent that either stimulates or inhibits the activity of PGC-3. The therapeutic agents of the invention have utility in the regulation of metabolism; in particular in obesity and the control of insulin resistance syndrome and other related disorders such as non-insulin dependent diabetes mellitus, dyslipidemia, and atherosclerosis.

In a further aspect of the invention we provide a screen for identifying compounds which modulate the activity of PGC-3, the invention extends to such a screen and to the use of compounds obtainable therefrom to modulate the activity of PGC-3 in vivo.

Potential therapeutic agents which may be tested in the screen include simple organic molecules, commonly known as “small molecules”, for example those having a molecular weight of less than 2000 Daltons. The screen may also be used to screen compound libraries such as peptide libraries, including synthetic peptide libraries and peptide phage libraries. Other suitable molecules include antibodies, nucleotide sequences and any other molecules which modulate the activity of PGC-3.

Once an inhibitor or stimulator of PGC-3 activity is identified then medicinal chemistry techniques can be applied to further refine its properties, for example to enhance efficacy and/or reduce side effects.

It will be appreciated that there are many screening procedures which may be employed to perform the present invention. Examples of suitable screening procedures which may be used to identify a PGC-3 modulator for use in the regulation of metabolism include rapid filtration of equilibrium binding mixtures, enzyme linked immunosorbent assays (ELISA), radioimmunoassays (RIA) and fluorescence resonance energy transfer assays (FRET). For further information on FRET the reader is directed to International Patent Application WO 94/28166 (Zeneca). Methods to identify potential drug candidates have been reviewed by Bevan P et al., 1995, TIBTECH 13 115.

A preferred method for identifying a compound capable of modulating the activity of PGC-3 is a scintillation proximity assay (SPA). SPA involves the use of fluomicrospheres coated with acceptor molecules, such as receptors, to which a ligand will bind selectively in a reversible manner (N Bosworth & P Towers, Nature, 341, 167-168, 1989). The technique requires the use of a ligand labelled with an isotope that emits low energy radiation which is dissipated easily into an aqueous medium. At any point during an assay, bound labelled ligands will be in close proximity to the fluomicrospheres, allowing the emitted energy to activate the fluor and produce light. In contrast, the vast majority of unbound labelled ligands will be too far from the fluomicrospheres to enable the transfer of energy. Bound ligands produce light but free ligands do not, allowing the extent of ligand binding to be measured without the need to separate bound and free ligand.

Cellular assay systems may be used to further identify PGC-3 modulators for use in the regulation of metabolism.

Therefore in a further aspect of the invention the candidate compound modulator is contacted with a host-cell which expresses an PGC-3 polypeptide (as hereindefined).

A preferred cellular assay system for use in the method of the invention is a two-hybrid assay system. The two-hybrid system utilises the ability of a pair of interacting proteins to bring the activation domain of a transcription factor into close proximity with its DNA-binding domain, restoring the functional activity of the transcription factor and inducing the expression of a reporter gene (S Fields & O Song, Nature, 340, 245-246, 1989). Commercially available systems such as the Clontech Matchmakers systems and protocols may be used with the present invention.

Other preferred cellular assay systems include measurement of changes in the levels of intracellular signalling molecules such as cyclic-AMP, intracellular calcium ions, or arachidonic acid metabolite release. These may all be measured using standard published procedures and commercially available reagents. In addition the polynucleotides of the present invention may be transfected into appropriate cell lines that have been transfected with a “reporter” gene such as bacterial lacZ, luciferase, aequorin or green fluorescent protein that will “report” these intracellular changes (Egerton et al, J. Mol, Endocrinol, 1995, 14(2), 179-189).

In a further aspect of the present invention we provide a novel PGC-3 modulator, or a pharmaceutically acceptable salt thereof, for use in a method of treatment of metabolic diseases of the human or animal body by therapy.

Examples of metabolic diseases which may be treated using a compound of the invention include insulin resistance syndrome, non-insulin dependent diabetes mellitus, dyslipidemia, obesity and atherosclerosis.

According to a further aspect of the invention, we provide a pharmaceutical composition which comprises a novel PGC-3 modulator, or a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable diluent or carrier.

The composition may be in the form suitable for oral use, for example a tablet, capsule, aqueous or oily solution, suspension or emulsion; for topical use, for example a cream, ointment, gel or an aqueous or oily solution or suspension; for nasal use, for example a snuff, nasal spray or nasal drops; for rectal use, for example a suppository; for administration by inhalation, for example as a finely divided powder such as a dry powder, a microcrystalline form or a liquid aerosol; for sub-lingual or buccal use, for example a tablet or capsule; or for parenteral use (including intravenous, subcutaneous, intramuscular, intravascular or infusion), for example a sterile aqueous or oily solution or suspension. In general, the above compositions may be prepared in a conventional manner using conventional excipients.

The invention also provides a method of treating a metabolic disease or medical condition mediated alone or in part by PGC-3, which comprises administering to a warm-blooded animal requiring such treatment an effective amount of an PGC-3 modulator as defined above.

The invention also provides the use of an PGC-3 modulator in the production of a medicament for use in the treatment of a metabolic disease.

The amount of active ingredient that is combined with one or more excipients to produce a single dosage form will necessarily vary depending on the subject treated and the particular route of administration. For example, a formulation intended for oral administration to humans will generally contain for example, from 0.5 mg to 2 g of active agent compounded with an appropriate and convenient amount of excipients which may vary from about 5 to about 98 percent by weight of the total composition. Dosage unit forms will generally contain about 1 mg to about 500 mg of an active ingredient.

The size of the dose for therapeutic or prophylactic purposes of an PGC-3 modulator will naturally vary according to the nature and severity of the immune disease, the age and sex of the patient, and the route of administration, according to well known principles of medicine.

In using an PGC-3 modulator for therapeutic or prophylactic purposes it will generally be administered so that a daily dose in the range for example 0.5 mg to 75 mg per kg body weight is received, given if required in divided doses. In general lower doses will be administered when a parenteral route is employed. Thus for example, for intravenous administration, a dose in the range for example 0.5 mg to 30 mg per kg body weight will generally be used. Similarly, for administration by inhalation a dose in the range for example 0.5 mg to 25 mg per kg body weight will be used.

The invention will now be illustrated but not limited by reference to the following Tables, Examples and Figures. Unless indicated otherwise, the techniques used are those detailed in well known molecular biology textbooks such as Sambrook, Fritsch & Maniatis, Molecular Cloning a Laboratory Manual, second edition, 1989, Cold Spring Harbor Laboratory Press.

SEQ ID NO.1 shows the full length human PGC-3a cDNA

SEQ ID NO.2.shows human PGC-3a protein sequence

SEQ ID NO.3.shows human PGC-3b cDNA

SEQ ID NO.4. shows human PGC-3b protein sequence

SEQ ID NO.5. shows the sequence of the 3′ RACE product isolated from human adipocyte cDNA.

SEQ ID NO.6. shows the sequence of the 5′RACE product isolated from human heart cDNA.

SEQ ID NO.7. shows human PGC-3c cDNA

SEQ ID NO 8 shows human PGC-3c protein sequence

SEQ ID NO 9 shows the full length rat PGC-3 cDNA

SEQ ID NO 10 shows rat PGC-3 protein sequence

FIGURE LEGENDS

FIG. 1 shows specific PCR products of 561 bp for PGC-3b and 491 bp for PGC-3a, isolated from breast adipose tissue cDNA, in [0092] lanes 1 and 2 respectively. The PGC-3b specific PCR product was obtained using PCR primers CME9830 and CME9831 (Table 2). The PGC-3a specific PCR product was obtained using PCR primers CME9830 and CME9850 (Table 2).
FIG. 2 shows a comparison of PGC-3a and PGC-3b with PGC-1, indicating that the molecules share regions of sequence homology in particular locations which are believed to be important for biological activity. [0093]
FIG. 3 shows a comparison of human PGC-3a and rat PGC-3 protein sequences indicating that the molecules share a high degree of sequence homology. [0094]
FIG. 4 shows the mean relative expression of PGC-3 mRNA in a range of human tissues. Quantitative real-time PCR was undertaken in quadruplicate on cDNA samples derived from the human tissues listed using Taqman™ fluorescent PCR technology (PE. Applied Biosystems). The normalised ratio of expression of PGC-3 relative to a housekeeping gene (GAPDH) was calculated for all tissues. SC=subcutaneous. Where multiple samples of the same tissue type were assayed the sample number is given, eg Omental adipocyte S24 refers to the data obtained from the omental [0095] adipocyte sample number 24. AU=arbitrary units.

TABLES

TABLE 1


Examples of conservative amino acid substitutions

	Original residue	Example conservative substitutions

	Ala (A)	Gly; Ser; Val; Leu; Ile; Pro
	Arg (R)	Lys; His; Gln; Asn
	Asn (N)	Gln; His; Lys; Arg
	Asp (D)	Glu
	Cys (C)	Ser
	Gln (Q)	Asn
	Glu (E)	Asp
	Gly (G)	Ala; Pro
	His (H)	Asn; Gln; Arg; Lys
	Ile (I)	Leu; Val; Met; Ala; Phe
	Leu (L)	Ile; Val; Met; Ala; Phe
	Lys (K)	Arg; Gln; His; Asn
	Met (M)	Leu; Tyr; Ile; Phe
	Phe (F)	Met; Leu; Tyr; Val; Ile; Ala
	Pro (P)	Ala; Gly
	Ser (S)	Thr
	Thr (T)	Ser
	Trp (W)	Tyr; Phe
	Tyr (Y)	Trp; Phe; Thr; Ser
	Val (V)	Ile; Leu; Met; Phe; Ala

TABLE 2


Primer sequences

Primer	Sequence

forward primer CME 9748	5′ GTCACAAAGCGACCCAACTT 3′

reverse primer CME 9749	5′ GAGTCATGGTCTCCAAAGGAAC 3′

AP1 adaptor primer	5′ CCATCCTAATACGACTCACTATAGGGC 3′

CME 9830 forward primer	5′ GCCACTCGAAGGAACTTCAGAT 3′

CME 9850 reverse primer B	5′ GGGTTAAGGCTGTTATCAATGC 3′

CME 9831 reverse primer A	5′ AGGCCAGAAGAGAAACAGGATG 3′

CME 9726 sequencing primer	5′ CTTCTCCTGTTCCTTTGGAGAC 3′

CME 9727 sequencing primer	5′ TGGGGTTCACTTGAGGATTG 3′

CME 9778 sequencing primer	5′ ATTCAAAATCTCTTCCAGCGAC 3′

CME 9776 sequencing primer	5′ GAAGACAGAAGCTGTGATGCTG 3′

TABLE 3


Primers used in Example 4

Primer	Sequence

SP1A

	5′-CATCACAGAGCACGTCTTGAG-3′

SP2A
	5′-CATGTAGCGTATGAGTTGCACCATC-3′

Oligo d(T)-anchor	5′-GACCACGCGTATCGATGTCGACTTTTT
primer	TTTTTTTTTTTV-3′
	V = A, C or G

PCR anchor primer	5′-GACCACGCGTATCGATGTCGAC-3

TABLE 4


Details of primers used to sequence rat PGC-3

	Primer	primer sequence (5′→3′)

	CVGI169	TTGGGTAACGCCAGGGTTTTCCCAGTCAC

	CVGI170	CCCCAGGCTTTACACTTTATGCTTCCGGC

	CVGI171	GCCAGTACAGCCCTGATGAT

	CVGI172	TCCCCAGTGTCTGAAGTGGATG

	CVGI281	CTCATTCGCTACATGCATACCT

	CVGI282	CGGCCTTGTGTCAAGGTGGATG

	CVGI283	CTTCTGGACTGAGTTCTCCATC

	CVGI390	CAGGAGACTGAATCCAGAGCTG

	CVGI391	GACAGTAGTCAAGGCCAGCAGC

	CVGI457	GAGACCATGACTACTGCCAGGT

	CVGI458	ACCGCTCTGGAGGAGGAAGACT

	CVGI535	TTAAGCCTTAACCCTTTGAGGA

	CVGI536	GGCCCAGATACACCGACTATGA

EXAMPLES

Example 1

Isolation of partial PGC-3 cDNA

Method [0100]
PCR primers CME 9748 and CME 9749, listed in Table 2 were synthesised and used to amplify a 347 bp product from human adipose cDNA (Human adipocyte Marathon Ready cDNA, Clontech Cat.# 7447-1, Clontech, Basingstoke, UK). [0101]
A technique known as Rapid Amplification of cDNA Ends (RACE) was then used to amplify the 3′ end of the PGC-3 cDNA. RACE is a commonly used molecular biological technique which enables the user to extend and identify sequence along a cDNA template in one direction. This allows the user to obtain a complete cDNA sequence starting from a small piece of cDNA sequence. For a more complete description of the method refer to Chenchick A, Moqadam F and Siebert P. 1996 Laboratory guide to RNA: isolation, analysis and synthesis. Wiley-Liss Inc. p273-321. In this case we used a commercially available RACE PCR kit, the Human adipocyte Marathon Ready™ cDNA (Clontech, Basingstoke, UK). It is a premade human adipocyte “library” of adaptor-ligated double stranded cDNA ready for performing both 5′ and 3′ RACE from the same template. The PGC-3 gene specific primer CME 9748 (Table 2) was used in a Marathon RACE reaction with the AP1 adapter primer (Table 2) supplied by Clontech and the Marathon Ready™ cDNA according to the manufacturer's instructions. [0102]
Results [0103]
A 1.5 kb PCR product was amplified in the reaction and separated from non-specific DNA by agarose gel electrophoresis using a 1.5% agarose gel and visualised by ethidium bromide staining. The 1.5 kb PCR product was isolated from the gel using a DNA extraction kit (Qiaex II™, Qiagen) and the purified PCR product cloned into PCR2.1™ vector using a TOPO TA™ Cloning kit (Invitrogen), according to the manufacturer's instructions. The cloned PCR product was fully sequenced using the vector M13 sequencing primers supplied in the kit (Invitrogen) and the PGC-3 gene specific sequencing primers CME 9726, CME 9727,CME 9778 and CME 9776 (listed in Table 2). The sequence of the 3′RACE product is shown in FIG. 5 (SEQ ID NO: 5). The predicted protein sequence of the 3′ end of SEQ ID NO:7 was found to be 50% identical to the sequence for human PGC-1 over a 135 aa region (see FIG. 7). This small region of SEQ ID NO:7 also shared 53% identity to the rat PGC-1 sequence (EMBL accession number: AB025784). [0104]
Two variants of PGC-3 have been found with cDNA sequence which differ at the 3′ end. We have named these two variants PGC-3a and PGC-3b. [0105]

Example 2

Isolation of a Full Length PGC-3a Clone From a Human Heart cDNA Library

Method [0106]
Primers CME 9830 and CME 9850 (Table 2) were synthesised based on the PGC-[0107] 3a 3′ RACE product sequence. These were used to PCR screen the Origene human heart cDNA library master plate (ORIGENE LHT-1001, Origene, USA) according to the manufacturers instructions.
Results [0108]
PCR screening of the master plate identified several wells positive for PGC-3a cDNA. The subplates corresponding to these wells were obtained from Origene and a subsequent round of PCR screening was performed to identify individual clones containing PGC-3a cDNA. [0109]
Clones containing PGC-3a were identified and sequenced. This resulted in the isolation of the complete cDNA sequence for PGC-3a (SEQ ID NO:1). The PGC-3a cDNA sequence comprises a coding region of 3009 nucleotides, that encode a protein of 1002 amino acids with a calculated molecular mass of 110 kDa and an estimated isoelectric point of 4.933. The protein sequence for PGC-3a is shown in SEQ ID NO:2. [0110]

Example 3

PCR Amplification of PGC-3a and PGC-3b From Human Breast Adipocyte cDNA

Methods [0111]
PCR primers were synthesised which would specifically amplify either PGC-3a or PGC-3b (CME 9830, CME 9831, CME 9850, Table 2). [0112]
To investigate whether both PGC-3a and PGC-3b were expressed by human breast adipocytes, PCRs were carried out using the above primers to amplify PGC-3a and PGC-3b from human breast adipocyte cDNA. The PCR conditions used were 94° C. for 1 minute then 30 cycles of 94° C. for 30 sec, then 68° C. for 4 minutes. The DNA polymerase used was Extensor™ from Advanced Biotechnologies. PCR was performed according to standard procedure described in Molecular Cloning,a laboratory manual, Sambrook, Fritsch and Maniatis Second Ed 1989). The forward PCR primer (CME 9830) was used in combination with either reverse PCR primer A (CME 9831) designed specifically to amplify PGC-3b (sequence 2) or reverse primer B (CME 9850) designed specifically to amplify [0113] sequence 3 PGC-3a (3′ RACE product).
Results [0114]
Specific PCR products of 561 bp for PGC-3b (CME9830/CME 9831) and 491 bp for PGC-3a (CME9830/CME9850) were obtained, as shown in FIG. 1 in [0115] lanes 1 and 2 respectively.
The complete cDNA sequence for PGC-3b is shown in SEQ ID NO:3. The PGC-3b cDNA sequence comprises a coding region of 2991 nucleotides encoding a protein of 996 amino acids. The protein sequence for PGC-3b is shown in SEQ ID NO:4. [0116]

Example 4

Isolation of partial PGC-3c cDNA

The technique known as RACE as described in example 1 was used to amplify the 5′ end of the PGC-3c cDNA. This procedure was undertaken using the [0117] Roche 5′/3 ′RACE kit (Cat. No. 1 734 792) and followed the manufacturer's instructions. First strand cDNA was synthesized from total human heart RNA (Stratagene, Cat. No. 73501241 ) using a gene specific primer (SP1A, listed in Table 3), AMV reverse transcriptase (supplied in kit) and deoxynucleotide mix (supplied in kit). The first strand cDNA was purified using High Pure PCR Product Purification Kit (Roche Diagnostics Corporation, Indianapolis, Ind., USA—Cat No. 1 732 668) according to the manufacturer's instructions. A homopolymeric A-tail was then added to the 3′end of the cDNA using terminate transferase using reagents and instructions supplied with the kit. The cDNA was then amplified by PCR using a gene specific primer (SP2A, see table 3) and an oligo dT-anchor primer (see table 3). The obtained cDNA was further amplified by a second PCR using a nested specific primer (SP3A, see Table 3) and a PCR anchor primer (see table 3). Resulting 5′RACE products were cloned into a vector. The cloned PCR products were fully sequenced. The sequence of the 5′RACE product is shown in Figure SEQ ID NO. 6. The full length cDNA sequence of PGC-3c is shown in SEQ ID NO. 7. The predicted protein sequence of PGC-3c is shown in SEQ ID NO. 8.

Example 5

Isolation of Full Length Rat PGC-3 cDNA

Homology searching using the human PGC-3 cDNA sequence identified a rat clone in a proprietary database that had a high level of homology to PGC-3. This clone was obtained and sequenced using primers CVGI169, CVGI170, CVGI171, CVGI172, CVGI281, CVGI282, CVGI283, CVGI390, CVGI391, CVGI457, CVGI458, CVGI535 (see table 4 for sequence information). The full length rat PGC-3 cDNA sequence is shown in SEQ ID NO. 9. The predicted protein sequence of rat PGC-3 is shown in Figure SEQ ID NO. 10. FIG. 13 shows a comparison of human PGC-3a and rat PGC-3 protein sequences, indicating that the molecules have a high degree of sequence homology (greater than 78% identity). [0118]

Example 6

Comparison of Human PGC-3 mRNA Expression Between Tissues

Total RNA was extracted from human adipocytes using TRI reagent (Sigma-Aldrich) following the manufacturer's suggested protocol. Two micrograms of total RNA from each adipocyte samples was used to generate cDNA, using the Promega reverse transcription system (Promega; catalogue number A3500) according to the manufacturer's instructions. The heart, skeletal muscle, kidney, liver, lung, heart and pancreas cDNAs were obtained from Clontech (Clontech, catalogue numbers K1420-1 and K1421-1). Probe and primer sequences were designed for PGC-3 and were: PGC-3 forward primer; 5′-TGCTGGCCCAGATACACTGA-3′. PGC-3 reverse primer; 5′-GGCTGTTATCAATGCAGGCTC-3′. PGC-3 probe; 5-FAM-CGTCAGGGAAAAGCAAGTATGAAGCCAT-TAMRA-3′. Taqman PCR assays for each target gene were performed in quadruplicate in 96 well plates on an ABI Prism 7700 Sequence Detection system (PE Applied Biosystems). For each 25 □1 Taqman reaction 0.01-1 ng cDNA was mixed with final concentrations of 1×Taqman Universal PCR Mastermix (PE Applied Biosystems), 300 nM forward and reverse primers and 200 nM probe. PCR parameters were 50° C. for 2 minutes, 95° C. for 10 minutes, 40 cycles of 95° C. for 15 seconds and 60° C. for 1 min. Results were analysed by the comparative Ct method as previously described in ABI Prism 7700 User Bulletin #2 (PE Applied Biosystems). Briefly, for each PCR, a threshold cycle (Ct) was calculated. This refers to the PCR cycle where amplified DNA is detectable above an arbitary threshold. Ct values are semi-quantitative and are related to the amount of target sequence present within a particular cDNA sample. Quadruplicate Ct samples were averaged and normalised by dividing with Ct values obtained for a housekeeping gene (GAPDH; supplied by PE Applied Biosystems). This gives a mean □Ct value for each cDNA. These values can be directly compared between different cDNA samples to give relative expression values for each target gene. FIG. 4 shows the mean relative expression of PGC-3 mRNA in the human tissues listed above. Where multiple samples of the same tissue type were assayed the sample number is given, eg Omental adipocyte S24 refers to the data obtained from the omental [0119] adipocyte sample number 24. These results demonstrate that PGC-3 is highly expressed in human breast adipocytes, omental adipocytes and subcutaneous adipocytes. High levels of expression of PGC-3 were also observed in lung and heart samples. Expression of PGC-3 was lower in human kidney, skeletal muscle, pancreas and liver samples.
1 40 1 3203 DNA Homo sapiens 1 ggcacgagga agaattgaac tcatacagct gatgggagtg tacaaaggtg gagggtccgg 60 ggaggagcaa ctctatgctg actttccaga acttgacctc tcccagctgg atgccagcga 120 ctttgactcg gccacctgct ttggggagct gcagtggtgc ccagagaact cagagactga 180 acccaaccag tacagccccg atgactccga gctcttccag attgacagtg agaatgaggc 240 cctcctggca gagctcacca agaccctgga tgacatccct gaagatgacg tgggtctggc 300 tgccttccca gccctggatg gtggagacgc tctatcatgc acctcagctt cgcctgcccc 360 ctcatctgca ccccccagcc ctgccccgga gaagccctcg gccccagccc ctgaggtgga 420 cgagctctca ctgctgcaga agctcctcct ggccacatcc tacccaacat caagctctga 480 cacccagaag gaagggaccg cctggcgcca ggcaggcctc agatctaaaa gtcaacggcc 540 ttgtgttaag gcggacagca cccaagacaa gaaggctccc atgatgcagt ctcagagccg 600 aagttgtaca gaactacata agcacctcac ctcggcacag tgctgcctgc aggatcgggg 660 tctgcagcca ccatgcctcc agagtccccg gctccctgcc aaggaggaca aggagccggg 720 tgaggactgc ccgagccccc agccagctcc agcctctccc caggactccc tagctctggg 780 cagggcagac cccggtgccc cggtttccca ggaagacatg caggcgatgg tgcaactcat 840 acgctacatg cacacctact gcctccccca gaggaagctg cccccacaga cccctgagcc 900 actccccaag gcctgcagca acccctccca gcaggtcaga tcccggccct ggtcccggca 960 ccactccaaa gcctcctggg ctgagttctc cattctgagg gaacttctgg ctcaagacgt 1020 gctctgtgat gtcagcaaac cctaccgtct ggccacgcct gtttatgcct ccctcacacc 1080 tcggtcaagg cccaggcccc ccaaagacag tcaggcctcc cctggtcgcc cgtcctcggt 1140 ggaggaggta aggatcgcag cttcacccaa gagcaccggg cccagaccaa gcctgcgccc 1200 actgcggctg gaggtgaaaa gggaggtccg ccggcctgcc agactgcagc agcaggagga 1260 ggaagacgag gaagaagagg aggaggaaga ggaagaagaa aaagaggagg aggaggagtg 1320 gggcaggaaa aggccaggcc gaggcctgcc atggacgaag ctggggagga agctggagag 1380 ctctgtgtgc cccgtgcggc gttctcggag actgaaccct gagctgggcc cctggctgac 1440 atttgcagat gagccgctgg tcccctcgga gccccaaggt gctctgccct cactgtgcct 1500 ggctcccaag gcctacgacg tagagcggga gctgggcagc cccacggacg aggacagtgg 1560 ccaagaccag cagctcctac ggggacccca gatccctgcc ctggagagcc cctgtgagag 1620 tgggtgtggg gacatggatg aggaccccag ctgcccgcag ctccctccca gagactctcc 1680 caggtgcctc atgctggcct tgtcacaaag cgacccaact tttggcaaga agagctttga 1740 gcagaccttg acagtggagc tctgtggcac agcaggactc accccaccca ccacaccacc 1800 gtacaagccc acagaggagg atcccttcaa accagacatc aagcatagtc taggcaaaga 1860 aatagctctc agcctcccct cccctgaggg cctctcactc aaggccaccc caggggctgc 1920 ccacaagctg ccaaagaagc acccagagcg aagtgagctc ctgtcccacc tgcgacatgc 1980 cacagcccag ccagcctccc aggctggcca gaagcgtccc ttctcctgtt cctttggaga 2040 ccatgactac tgccaggtgc tccgaccaga aggcgtcctg caaaggaagg tgctgaggtc 2100 ctgggagccg tctggggttc accttgagga ctggccccag cagggtgccc cttgggctga 2160 ggcacaggcc cctggcaggg aggaagacag aagctgtgat gctggtgccc cacccaagga 2220 cagcacgctg ctgagagacc atgagatccg tgctagcctc accaaacact ttgggctgct 2280 ggagaccgcc ctggaggagg aagacctggc ctcctgcaag agccctgagt atgacactgt 2340 ctttgaagac agcagcagca gcagcggcga gagcagcttc ctcccagagg aggaagagga 2400 agaaggggag gaggaggagg aggacgatga agaagaggac tcaggggtca gccccacttg 2460 ctctgaccac tgcccctacc agagcccacc aagcaaggcc aaccggcagc tctgttcccg 2520 cagccgctca agctctggct cttcaccctg ccactcctgg tcaccagcca ctcgaaggaa 2580 cttcagatgt gagagcagag ggccgtgttc agacagaacg ccaagcatcc ggcacgccag 2640 gaagcggcgg gaaaaggcca ttggggaagg ccgcgtggtg tacattcaaa atctctccag 2700 cgacatgagc tcccgagagc tgaagaggcg ctttgaagtg tttggtgaga ttgaggagtg 2760 cgaggtgctg acaagaaata ggagaggcga gaagtacggc ttcatcacct accggtgttc 2820 tgagcacgcg gccctctctt tgacaaaggg cgctgccctg aggaagcgca acgagccctc 2880 cttccagctg agctacggag ggctccggca cttctgctgg cccagataca ctgactacga 2940 ttccaattca gaagaggccc ttcctgcgtc agggaaaagc aagtatgaag ccatggattt 3000 tgacagctta ctgaaagagg cccagcagag cctgcattga taacagcctt aaccctcgag 3060 gaatacctca atacctcaga caaggccctt ccaatatgtt tacgttttca aagaaatcaa 3120 gtatatgagg agagcgagcg agcgtgagag aacacccgtg agagagactt gaaactgctg 3180 tcctttaaaa aaaaaaaaaa aaa 3203 2 1002 PRT Homo sapiens 2 Met Gly Val Tyr Lys Gly Gly Gly Ser Gly Glu Glu Gln Leu Tyr Ala 1 5 10 15 Asp Phe Pro Glu Leu Asp Leu Ser Gln Leu Asp Ala Ser Asp Phe Asp 20 25 30 Ser Ala Thr Cys Phe Gly Glu Leu Gln Trp Cys Pro Glu Asn Ser Glu 35 40 45 Thr Glu Pro Asn Gln Tyr Ser Pro Asp Asp Ser Glu Leu Phe Gln Ile 50 55 60 Asp Ser Glu Asn Glu Ala Leu Leu Ala Glu Leu Thr Lys Thr Leu Asp 65 70 75 80 Asp Ile Pro Glu Asp Asp Val Gly Leu Ala Ala Phe Pro Ala Leu Asp 85 90 95 Gly Gly Asp Ala Leu Ser Cys Thr Ser Ala Ser Pro Ala Pro Ser Ser 100 105 110 Ala Pro Pro Ser Pro Ala Pro Glu Lys Pro Ser Ala Pro Ala Pro Glu 115 120 125 Val Asp Glu Leu Ser Leu Leu Gln Lys Leu Leu Leu Ala Thr Ser Tyr 130 135 140 Pro Thr Ser Ser Ser Asp Thr Gln Lys Glu Gly Thr Ala Trp Arg Gln 145 150 155 160 Ala Gly Leu Arg Ser Lys Ser Gln Arg Pro Cys Val Lys Ala Asp Ser 165 170 175 Thr Gln Asp Lys Lys Ala Pro Met Met Gln Ser Gln Ser Arg Ser Cys 180 185 190 Thr Glu Leu His Lys His Leu Thr Ser Ala Gln Cys Cys Leu Gln Asp 195 200 205 Arg Gly Leu Gln Pro Pro Cys Leu Gln Ser Pro Arg Leu Pro Ala Lys 210 215 220 Glu Asp Lys Glu Pro Gly Glu Asp Cys Pro Ser Pro Gln Pro Ala Pro 225 230 235 240 Ala Ser Pro Gln Asp Ser Leu Ala Leu Gly Arg Ala Asp Pro Gly Ala 245 250 255 Pro Val Ser Gln Glu Asp Met Gln Ala Met Val Gln Leu Ile Arg Tyr 260 265 270 Met His Thr Tyr Cys Leu Pro Gln Arg Lys Leu Pro Pro Gln Thr Pro 275 280 285 Glu Pro Leu Pro Lys Ala Cys Ser Asn Pro Ser Gln Gln Val Arg Ser 290 295 300 Arg Pro Trp Ser Arg His His Ser Lys Ala Ser Trp Ala Glu Phe Ser 305 310 315 320 Ile Leu Arg Glu Leu Leu Ala Gln Asp Val Leu Cys Asp Val Ser Lys 325 330 335 Pro Tyr Arg Leu Ala Thr Pro Val Tyr Ala Ser Leu Thr Pro Arg Ser 340 345 350 Arg Pro Arg Pro Pro Lys Asp Ser Gln Ala Ser Pro Gly Arg Pro Ser 355 360 365 Ser Val Glu Glu Val Arg Ile Ala Ala Ser Pro Lys Ser Thr Gly Pro 370 375 380 Arg Pro Ser Leu Arg Pro Leu Arg Leu Glu Val Lys Arg Glu Val Arg 385 390 395 400 Arg Pro Ala Arg Leu Gln Gln Gln Glu Glu Glu Asp Glu Glu Glu Glu 405 410 415 Glu Glu Glu Glu Glu Glu Glu Lys Glu Glu Glu Glu Glu Trp Gly Arg 420 425 430 Lys Arg Pro Gly Arg Gly Leu Pro Trp Thr Lys Leu Gly Arg Lys Leu 435 440 445 Glu Ser Ser Val Cys Pro Val Arg Arg Ser Arg Arg Leu Asn Pro Glu 450 455 460 Leu Gly Pro Trp Leu Thr Phe Ala Asp Glu Pro Leu Val Pro Ser Glu 465 470 475 480 Pro Gln Gly Ala Leu Pro Ser Leu Cys Leu Ala Pro Lys Ala Tyr Asp 485 490 495 Val Glu Arg Glu Leu Gly Ser Pro Thr Asp Glu Asp Ser Gly Gln Asp 500 505 510 Gln Gln Leu Leu Arg Gly Pro Gln Ile Pro Ala Leu Glu Ser Pro Cys 515 520 525 Glu Ser Gly Cys Gly Asp Met Asp Glu Asp Pro Ser Cys Pro Gln Leu 530 535 540 Pro Pro Arg Asp Ser Pro Arg Cys Leu Met Leu Ala Leu Ser Gln Ser 545 550 555 560 Asp Pro Thr Phe Gly Lys Lys Ser Phe Glu Gln Thr Leu Thr Val Glu 565 570 575 Leu Cys Gly Thr Ala Gly Leu Thr Pro Pro Thr Thr Pro Pro Tyr Lys 580 585 590 Pro Thr Glu Glu Asp Pro Phe Lys Pro Asp Ile Lys His Ser Leu Gly 595 600 605 Lys Glu Ile Ala Leu Ser Leu Pro Ser Pro Glu Gly Leu Ser Leu Lys 610 615 620 Ala Thr Pro Gly Ala Ala His Lys Leu Pro Lys Lys His Pro Glu Arg 625 630 635 640 Ser Glu Leu Leu Ser His Leu Arg His Ala Thr Ala Gln Pro Ala Ser 645 650 655 Gln Ala Gly Gln Lys Arg Pro Phe Ser Cys Ser Phe Gly Asp His Asp 660 665 670 Tyr Cys Gln Val Leu Arg Pro Glu Gly Val Leu Gln Arg Lys Val Leu 675 680 685 Arg Ser Trp Glu Pro Ser Gly Val His Leu Glu Asp Trp Pro Gln Gln 690 695 700 Gly Ala Pro Trp Ala Glu Ala Gln Ala Pro Gly Arg Glu Glu Asp Arg 705 710 715 720 Ser Cys Asp Ala Gly Ala Pro Pro Lys Asp Ser Thr Leu Leu Arg Asp 725 730 735 His Glu Ile Arg Ala Ser Leu Thr Lys His Phe Gly Leu Leu Glu Thr 740 745 750 Ala Leu Glu Glu Glu Asp Leu Ala Ser Cys Lys Ser Pro Glu Tyr Asp 755 760 765 Thr Val Phe Glu Asp Ser Ser Ser Ser Ser Gly Glu Ser Ser Phe Leu 770 775 780 Pro Glu Glu Glu Glu Glu Glu Gly Glu Glu Glu Glu Glu Asp Asp Glu 785 790 795 800 Glu Glu Asp Ser Gly Val Ser Pro Thr Cys Ser Asp His Cys Pro Tyr 805 810 815 Gln Ser Pro Pro Ser Lys Ala Asn Arg Gln Leu Cys Ser Arg Ser Arg 820 825 830 Ser Ser Ser Gly Ser Ser Pro Cys His Ser Trp Ser Pro Ala Thr Arg 835 840 845 Arg Asn Phe Arg Cys Glu Ser Arg Gly Pro Cys Ser Asp Arg Thr Pro 850 855 860 Ser Ile Arg His Ala Arg Lys Arg Arg Glu Lys Ala Ile Gly Glu Gly 865 870 875 880 Arg Val Val Tyr Ile Gln Asn Leu Ser Ser Asp Met Ser Ser Arg Glu 885 890 895 Leu Lys Arg Arg Phe Glu Val Phe Gly Glu Ile Glu Glu Cys Glu Val 900 905 910 Leu Thr Arg Asn Arg Arg Gly Glu Lys Tyr Gly Phe Ile Thr Tyr Arg 915 920 925 Cys Ser Glu His Ala Ala Leu Ser Leu Thr Lys Gly Ala Ala Leu Arg 930 935 940 Lys Arg Asn Glu Pro Ser Phe Gln Leu Ser Tyr Gly Gly Leu Arg His 945 950 955 960 Phe Cys Trp Pro Arg Tyr Thr Asp Tyr Asp Ser Asn Ser Glu Glu Ala 965 970 975 Leu Pro Ala Ser Gly Lys Ser Lys Tyr Glu Ala Met Asp Phe Asp Ser 980 985 990 Leu Leu Lys Glu Ala Gln Gln Ser Leu His 995 1000 3 3679 DNA Homo sapiens 3 ggcacgagga agaattgaac tcatacagct gatgggagtg tacaaaggtg gagggtccgg 60 ggaggagcaa ctctatgctg actttccaga acttgacctc tcccagctgg atgccagcga 120 ctttgactcg gccacctgct ttggggagct gcagtggtgc ccagagaact cagagactga 180 acccaaccag tacagccccg atgactccga gctcttccag attgacagtg agaatgaggc 240 cctcctggca gagctcacca agaccctgga tgacatccct gaagatgacg tgggtctggc 300 tgccttccca gccctggatg gtggagacgc tctatcatgc acctcagctt cgcctgcccc 360 ctcatctgca ccccccagcc ctgccccgga gaagccctcg gccccagccc ctgaggtgga 420 cgagctctca ctgctgcaga agctcctcct ggccacatcc tacccaacat caagctctga 480 cacccagaag gaagggaccg cctggcgcca ggcaggcctc agatctaaaa gtcaacggcc 540 ttgtgttaag gcggacagca cccaagacaa gaaggctccc atgatgcagt ctcagagccg 600 aagttgtaca gaactacata agcacctcac ctcggcacag tgctgcctgc aggatcgggg 660 tctgcagcca ccatgcctcc agagtccccg gctccctgcc aaggaggaca aggagccggg 720 tgaggactgc ccgagccccc agccagctcc agcctctccc caggactccc tagctctggg 780 cagggcagac cccggtgccc cggtttccca ggaagacatg caggcgatgg tgcaactcat 840 acgctacatg cacacctact gcctccccca gaggaagctg cccccacaga cccctgagcc 900 actccccaag gcctgcagca acccctccca gcaggtcaga tcccggccct ggtcccggca 960 ccactccaaa gcctcctggg ctgagttctc cattctgagg gaacttctgg ctcaagacgt 1020 gctctgtgat gtcagcaaac cctaccgtct ggccacgcct gtttatgcct ccctcacacc 1080 tcggtcaagg cccaggcccc ccaaagacag tcaggcctcc cctggtcgcc cgtcctcggt 1140 ggaggaggta aggatcgcag cttcacccaa gagcaccggg cccagaccaa gcctgcgccc 1200 actgcggctg gaggtgaaaa gggaggtccg ccggcctgcc agactgcagc agcaggagga 1260 ggaagacgag gaagaagagg aggaggaaga ggaagaagaa aaagaggagg aggaggagtg 1320 gggcaggaaa aggccaggcc gaggcctgcc atggacgaag ctggggagga agctggagag 1380 ctctgtgtgc cccgtgcggc gttctcggag actgaaccct gagctgggcc cctggctgac 1440 atttgcagat gagccgctgg tcccctcgga gccccaaggt gctctgccct cactgtgcct 1500 ggctcccaag gcctacgacg tagagcggga gctgggcagc cccacggacg aggacagtgg 1560 ccaagaccag cagctcctac ggggacccca gatccctgcc ctggagagcc cctgtgagag 1620 tgggtgtggg gacatggatg aggaccccag ctgcccgcag ctccctccca gagactctcc 1680 caggtgcctc atgctggcct tgtcacaaag cgacccaact tttggcaaga agagctttga 1740 gcagaccttg acagtggagc tctgtggcac agcaggactc accccaccca ccacaccacc 1800 gtacaagccc acagaggagg atcccttcaa accagacatc aagcatagtc taggcaaaga 1860 aatagctctc agcctcccct cccctgaggg cctctcactc aaggccaccc caggggctgc 1920 ccacaagctg ccaaagaagc acccagagcg aagtgagctc ctgtcccacc tgcgacatgc 1980 cacagcccag ccagcctccc aggctggcca gaagcgtccc ttctcctgtt cctttggaga 2040 ccatgactac tgccaggtgc tccgaccaga aggcgtcctg caaaggaagg tgctgaggtc 2100 ctgggagccg tctggggttc accttgagga ctggccccag cagggtgccc cttgggctga 2160 ggcacaggcc cctggcaggg aggaagacag aagctgtgat gctggcgccc cacccaagga 2220 cagcacgctg ctgagagacc atgagatccg tgccagcctc accaaacact ttgggctgct 2280 ggagaccgcc ctggaggagg aagacctggc ctcctgcaag agccctgagt atgacactgt 2340 ctttgaagac agcagcagca gcagcggcga gagcagcttc ctcccagagg aggaagagga 2400 agaaggggag gaggaggagg aggacgatga agaagaggac tcaggggtca gccccacttg 2460 ctctgaccac tgcccctacc agagcccacc aagcaaggcc aaccggcagc tctgttcccg 2520 cagccgctca agctctggct cttcaccctg ccactcctgg tcaccagcca ctcgaaggaa 2580 cttcagatgt gagagcagag ggccgtgttc agacagaacg ccaagcatcc ggcacgccag 2640 gaagcggcgg gaaaaggcca ttggggaagg ccgcgtggtg tacattcaaa atctctccag 2700 cgacatgagc tcccgagagc tgaagaggcg ctttgaagtg tttggtgaga ttgaggagtg 2760 cgaggtgctg acaagaaata ggagaggcga gaagtacggc ttcatcacct accggtgttc 2820 tgagcacgcg gccctctctt tgacaaaggg cgctgccctg aggaagcgca acgagccctc 2880 cttccagctg agctacggag ggctccggca cttctgctgg cccagataca ctgactacgg 2940 taagcccctg aaacccagcc acagtctagt aagactcaaa gcttgggaag cagtgccttc 3000 cttgaacaaa acccagagct aaagcgcctt gtggacatag cttccatccc cacaccccag 3060 tgtgctgctt ggtataactt tgcagccact ttgcctgaag actaccatcc tgtttctctt 3120 ctggcctctg gtccacctta tcctgtcctg tgactgctac caaagagaat ccagcctccc 3180 acggcctcta ggaagattca gtcatgtgca cagccagctg gcagaaccgt ggctacggtc 3240 tccttgactt cacagggcca gctgctaccc tgtccccttc aggggcattc cgtggtgacc 3300 ccagacaagg cagcagccac ctggggacaa gatgatgaag aaggacaaag aagtacaatg 3360 tacgaaagaa ttacttggcc aggctcagtg gctcatgcct gtaatcccat caccttggga 3420 ggctgaggca agaggatcac ttgagcccag gagttcgaga ccagcttggg caacatagtg 3480 aaatcctgtc tctacaaaaa atataaaaat tagccaggca tggtggcttg cgcctatagt 3540 cccagctact caggaggcag aggtgggagg atcacctgaa cccaagaggt tggagctgca 3600 gtgagccatg atggcactac tgcattccag cctgggcaac agagcaagac cctgtctcaa 3660 aaggaaaaaa aaaaaaaaa 3679 4 996 PRT Homo sapiens 4 Met Gly Val Tyr Lys Gly Gly Gly Ser Gly Glu Glu Gln Leu Tyr Ala 1 5 10 15 Asp Phe Pro Glu Leu Asp Leu Ser Gln Leu Asp Ala Ser Asp Phe Asp 20 25 30 Ser Ala Thr Cys Phe Gly Glu Leu Gln Trp Cys Pro Glu Asn Ser Glu 35 40 45 Thr Glu Pro Asn Gln Tyr Ser Pro Asp Asp Ser Glu Leu Phe Gln Ile 50 55 60 Asp Ser Glu Asn Glu Ala Leu Leu Ala Glu Leu Thr Lys Thr Leu Asp 65 70 75 80 Asp Ile Pro Glu Asp Asp Val Gly Leu Ala Ala Phe Pro Ala Leu Asp 85 90 95 Gly Gly Asp Ala Leu Ser Cys Thr Ser Ala Ser Pro Ala Pro Ser Ser 100 105 110 Ala Pro Pro Ser Pro Ala Pro Glu Lys Pro Ser Ala Pro Ala Pro Glu 115 120 125 Val Asp Glu Leu Ser Leu Leu Gln Lys Leu Leu Leu Ala Thr Ser Tyr 130 135 140 Pro Thr Ser Ser Ser Asp Thr Gln Lys Glu Gly Thr Ala Trp Arg Gln 145 150 155 160 Ala Gly Leu Arg Ser Lys Ser Gln Arg Pro Cys Val Lys Ala Asp Ser 165 170 175 Thr Gln Asp Lys Lys Ala Pro Met Met Gln Ser Gln Ser Arg Ser Cys 180 185 190 Thr Glu Leu His Lys His Leu Thr Ser Ala Gln Cys Cys Leu Gln Asp 195 200 205 Arg Gly Leu Gln Pro Pro Cys Leu Gln Ser Pro Arg Leu Pro Ala Lys 210 215 220 Glu Asp Lys Glu Pro Gly Glu Asp Cys Pro Ser Pro Gln Pro Ala Pro 225 230 235 240 Ala Ser Pro Gln Asp Ser Leu Ala Leu Gly Arg Ala Asp Pro Gly Ala 245 250 255 Pro Val Ser Gln Glu Asp Met Gln Ala Met Val Gln Leu Ile Arg Tyr 260 265 270 Met His Thr Tyr Cys Leu Pro Gln Arg Lys Leu Pro Pro Gln Thr Pro 275 280 285 Glu Pro Leu Pro Lys Ala Cys Ser Asn Pro Ser Gln Gln Val Arg Ser 290 295 300 Arg Pro Trp Ser Arg His His Ser Lys Ala Ser Trp Ala Glu Phe Ser 305 310 315 320 Ile Leu Arg Glu Leu Leu Ala Gln Asp Val Leu Cys Asp Val Ser Lys 325 330 335 Pro Tyr Arg Leu Ala Thr Pro Val Tyr Ala Ser Leu Thr Pro Arg Ser 340 345 350 Arg Pro Arg Pro Pro Lys Asp Ser Gln Ala Ser Pro Gly Arg Pro Ser 355 360 365 Ser Val Glu Glu Val Arg Ile Ala Ala Ser Pro Lys Ser Thr Gly Pro 370 375 380 Arg Pro Ser Leu Arg Pro Leu Arg Leu Glu Val Lys Arg Glu Val Arg 385 390 395 400 Arg Pro Ala Arg Leu Gln Gln Gln Glu Glu Glu Asp Glu Glu Glu Glu 405 410 415 Glu Glu Glu Glu Glu Glu Glu Lys Glu Glu Glu Glu Glu Trp Gly Arg 420 425 430 Lys Arg Pro Gly Arg Gly Leu Pro Trp Thr Lys Leu Gly Arg Lys Leu 435 440 445 Glu Ser Ser Val Cys Pro Val Arg Arg Ser Arg Arg Leu Asn Pro Glu 450 455 460 Leu Gly Pro Trp Leu Thr Phe Ala Asp Glu Pro Leu Val Pro Ser Glu 465 470 475 480 Pro Gln Gly Ala Leu Pro Ser Leu Cys Leu Ala Pro Lys Ala Tyr Asp 485 490 495 Val Glu Arg Glu Leu Gly Ser Pro Thr Asp Glu Asp Ser Gly Gln Asp 500 505 510 Gln Gln Leu Leu Arg Gly Pro Gln Ile Pro Ala Leu Glu Ser Pro Cys 515 520 525 Glu Ser Gly Cys Gly Asp Met Asp Glu Asp Pro Ser Cys Pro Gln Leu 530 535 540 Pro Pro Arg Asp Ser Pro Arg Cys Leu Met Leu Ala Leu Ser Gln Ser 545 550 555 560 Asp Pro Thr Phe Gly Lys Lys Ser Phe Glu Gln Thr Leu Thr Val Glu 565 570 575 Leu Cys Gly Thr Ala Gly Leu Thr Pro Pro Thr Thr Pro Pro Tyr Lys 580 585 590 Pro Thr Glu Glu Asp Pro Phe Lys Pro Asp Ile Lys His Ser Leu Gly 595 600 605 Lys Glu Ile Ala Leu Ser Leu Pro Ser Pro Glu Gly Leu Ser Leu Lys 610 615 620 Ala Thr Pro Gly Ala Ala His Lys Leu Pro Lys Lys His Pro Glu Arg 625 630 635 640 Ser Glu Leu Leu Ser His Leu Arg His Ala Thr Ala Gln Pro Ala Ser 645 650 655 Gln Ala Gly Gln Lys Arg Pro Phe Ser Cys Ser Phe Gly Asp His Asp 660 665 670 Tyr Cys Gln Val Leu Arg Pro Glu Gly Val Leu Gln Arg Lys Val Leu 675 680 685 Arg Ser Trp Glu Pro Ser Gly Val His Leu Glu Asp Trp Pro Gln Gln 690 695 700 Gly Ala Pro Trp Ala Glu Ala Gln Ala Pro Gly Arg Glu Glu Asp Arg 705 710 715 720 Ser Cys Asp Ala Gly Ala Pro Pro Lys Asp Ser Thr Leu Leu Arg Asp 725 730 735 His Glu Ile Arg Ala Ser Leu Thr Lys His Phe Gly Leu Leu Glu Thr 740 745 750 Ala Leu Glu Glu Glu Asp Leu Ala Ser Cys Lys Ser Pro Glu Tyr Asp 755 760 765 Thr Val Phe Glu Asp Ser Ser Ser Ser Ser Gly Glu Ser Ser Phe Leu 770 775 780 Pro Glu Glu Glu Glu Glu Glu Gly Glu Glu Glu Glu Glu Asp Asp Glu 785 790 795 800 Glu Glu Asp Ser Gly Val Ser Pro Thr Cys Ser Asp His Cys Pro Tyr 805 810 815 Gln Ser Pro Pro Ser Lys Ala Asn Arg Gln Leu Cys Ser Arg Ser Arg 820 825 830 Ser Ser Ser Gly Ser Ser Pro Cys His Ser Trp Ser Pro Ala Thr Arg 835 840 845 Arg Asn Phe Arg Cys Glu Ser Arg Gly Pro Cys Ser Asp Arg Thr Pro 850 855 860 Ser Ile Arg His Ala Arg Lys Arg Arg Glu Lys Ala Ile Gly Glu Gly 865 870 875 880 Arg Val Val Tyr Ile Gln Asn Leu Ser Ser Asp Met Ser Ser Arg Glu 885 890 895 Leu Lys Arg Arg Phe Glu Val Phe Gly Glu Ile Glu Glu Cys Glu Val 900 905 910 Leu Thr Arg Asn Arg Arg Gly Glu Lys Tyr Gly Phe Ile Thr Tyr Arg 915 920 925 Cys Ser Glu His Ala Ala Leu Ser Leu Thr Lys Gly Ala Ala Leu Arg 930 935 940 Lys Arg Asn Glu Pro Ser Phe Gln Leu Ser Tyr Gly Gly Leu Arg His 945 950 955 960 Phe Cys Trp Pro Arg Tyr Thr Asp Tyr Gly Lys Pro Leu Lys Pro Ser 965 970 975 His Ser Leu Val Arg Leu Lys Ala Trp Glu Ala Val Pro Ser Leu Asn 980 985 990 Lys Thr Gln Ser 995 5 1496 DNA Homo sapiens 5 gtcacaaagc gacccaactt ttggcaagaa gagctttgag cagaccttga cagtggagct 60 ctgtggcaca gcaggactca ccccacccac cacaccaccg tacaagccca cagaggagga 120 tcccttcaaa ccagacatca agcatagtct aggcaaagaa atagctctca gcctcccctc 180 ccctgagggc ctctcactca aggccacccc aggggctgcc cacaagctgc caaagaagca 240 cccagagcga agtgagctcc tgtcccacct gcgacatgcc acagcccagc cagcctccca 300 ggctggccag aagcgtccct tctcctgttc ctttggagac catgactact gccaggtgct 360 ccgaccagaa ggcgtcctgc aaaggaaggt gctgaggtcc tgggagccgt ctggggttca 420 ccttgaggac tggccccagc agggtgcccc ttgggctgag gcacaggccc ctggcaggga 480 ggaagacaga agctgtgatg ctggcgcccc acccaaggac agcacgctgc tgagagacca 540 tgagatccgt gccagcctca ccaaacactt tgggctgctg gagaccgccc tggaggagga 600 agacctggcc tcctgcaaga gccctgagta tgacactgtc tttgaagaca gcagcagcag 660 cagcggcgag agcagcttcc tcccagagga ggaagaggaa gaaggggagg aggaggagga 720 ggacgatgaa gaagaggact caggggtcag ccccacttgc tctgaccact gcccctacca 780 gagcccacca agcaaggcca accggcagct ctgttcccgc agccgctcaa gctctggctc 840 ttcaccctgc cactcctggt caccagccac tcgaaggaac ttcagcagat gtgagagcag 900 agggccgtgt tcagacagaa cgccaagcat ccggcacgcc aggaagcggc gggaaaaggc 960 cattggggaa ggccgcgtgg tgtacattca aaatctctcc agcgacatga gctcccgaga 1020 gctgaagagg cgctttgaag tgtttggtga gattgaggag tgcgaggtgc tgacaagaaa 1080 taggagaggc gagaagtacg gcttcatcac ctaccggtgt tctgagcacg cggccctctc 1140 tttgacaaag ggcgctgccc tgaggaagcg caacgagccc tccttccagc tgagctacgg 1200 agggctccgg cacttctgct ggcccagata cactgactac gattccaatt cagaagaggc 1260 ccttcctgcg tcagggaaaa gcaagtatga agccatggat tttgacagct tactgaaaga 1320 ggcccagcag agcctgcatt gataacagcc ttaaccctcg aggaatacct caatacctca 1380 gacaaggccc ttccaatatg tttacgtttt caaagaaatc aagtatatga ggagagcgag 1440 cgagcgtgag agaacacccg tgagagagac ttgaaactgc tgtcctaaaa aaaaaa 1496 6 172 DNA Homo sapiens 6 actccgccgc acgctgcagc cgcggctgga agatggcggg gaacgactgc ggcgcgctgc 60 tggacgaaga gctctcctcc ttcttcctca actatctcgc tgacacgcag ggtggagggt 120 ccggggagga gcaactctat gctgactttc cagaacttga cctctcccag ct 172 7 3267 DNA Homo sapiens 7 actccgccgc acgctgcagc cgcggctgga agatggcggg gaacgactgc ggcgcgctgc 60 tggacgaaga gctctcctcc ttcttcctca actatctcgc tgacacgcag ggtggagggt 120 ccggggagga gcaactctat gctgactttc cagaacttga cctctcccag ctggatgcca 180 gcgactttga ctcggccacc tgctttgggg agctgcagtg gtgcccagag aactcagaga 240 ctgaacccaa ccagtacagc cccgatgact ccgagctctt ccagattgac agtgagaatg 300 aggccctcct ggcagagctc accaagaccc tggatgacat ccctgaagat gacgtgggtc 360 tggctgcctt cccagccctg gatggtggag acgctctatc atgcacctca gcttcgcctg 420 ccccctcatc tgcacccccc agccctgccc cggagaagcc ctcggcccca gcccctgagg 480 tggacgagct ctcactgctg cagaagctcc tcctggccac atcctaccca acatcaagct 540 ctgacaccca gaaggaaggg accgcctggc gccaggcagg cctcagatct aaaagtcaac 600 ggccttgtgt taaggcggac agcacccaag acaagaaggc tcccatgatg cagtctcaga 660 gccgaagttg tacagaacta cataagcacc tcacctcggc acagtgctgc ctgcaggatc 720 ggggtctgca gccaccatgc ctccagagtc cccggctccc tgccaaggag gacaaggagc 780 cgggtgagga ctgcccgagc ccccagccag ctccagcctc tccccaggac tccctagctc 840 tgggcagggc agaccccggt gccccggttt cccaggaaga catgcaggcg atggtgcaac 900 tcatacgcta catgcacacc tactgcctcc cccagaggaa gctgccccca cagacccctg 960 agccactccc caaggcctgc agcaacccct cccagcaggt cagatcccgg ccctggtccc 1020 ggcaccactc caaagcctcc tgggctgagt tctccattct gagggaactt ctggctcaag 1080 acgtgctctg tgatgtcagc aaaccctacc gtctggccac gcctgtttat gcctccctca 1140 cacctcggtc aaggcccagg ccccccaaag acagtcaggc ctcccctggt cgcccgtcct 1200 cggtggagga ggtaaggatc gcagcttcac ccaagagcac cgggcccaga ccaagcctgc 1260 gcccactgcg gctggaggtg aaaagggagg tccgccggcc tgccagactg cagcagcagg 1320 aggaggaaga cgaggaagaa gaggaggagg aagaggaaga agaaaaagag gaggaggagg 1380 agtggggcag gaaaaggcca ggccgaggcc tgccatggac gaagctgggg aggaagctgg 1440 agagctctgt gtgccccgtg cggcgttctc ggagactgaa ccctgagctg ggcccctggc 1500 tgacatttgc agatgagccg ctggtcccct cggagcccca aggtgctctg ccctcactgt 1560 gcctggctcc caaggcctac gacgtagagc gggagctggg cagccccacg gacgaggaca 1620 gtggccaaga ccagcagctc ctacggggac cccagatccc tgccctggag agcccctgtg 1680 agagtgggtg tggggacatg gatgaggacc ccagctgccc gcagctccct cccagagact 1740 ctcccaggtg cctcatgctg gccttgtcac aaagcgaccc aacttttggc aagaagagct 1800 ttgagcagac cttgacagtg gagctctgtg gcacagcagg actcacccca cccaccacac 1860 caccgtacaa gcccacagag gaggatccct tcaaaccaga catcaagcat agtctaggca 1920 aagaaatagc tctcagcctc ccctcccctg agggcctctc actcaaggcc accccagggg 1980 ctgcccacaa gctgccaaag aagcacccag agcgaagtga gctcctgtcc cacctgcgac 2040 atgccacagc ccagccagcc tcccaggctg gccagaagcg tcccttctcc tgttcctttg 2100 gagaccatga ctactgccag gtgctccgac cagaaggcgt cctgcaaagg aaggtgctga 2160 ggtcctggga gccgtctggg gttcaccttg aggactggcc ccagcagggt gccccttggg 2220 ctgaggcaca ggcccctggc agggaggaag acagaagctg tgatgctggt gccccaccca 2280 aggacagcac gctgctgaga gaccatgaga tccgtgctag cctcaccaaa cactttgggc 2340 tgctggagac cgccctggag gaggaagacc tggcctcctg caagagccct gagtatgaca 2400 ctgtctttga agacagcagc agcagcagcg gcgagagcag cttcctccca gaggaggaag 2460 aggaagaagg ggaggaggag gaggaggacg atgaagaaga ggactcaggg gtcagcccca 2520 cttgctctga ccactgcccc taccagagcc caccaagcaa ggccaaccgg cagctctgtt 2580 cccgcagccg ctcaagctct ggctcttcac cctgccactc ctggtcacca gccactcgaa 2640 ggaacttcag atgtgagagc agagggccgt gttcagacag aacgccaagc atccggcacg 2700 ccaggaagcg gcgggaaaag gccattgggg aaggccgcgt ggtgtacatt caaaatctct 2760 ccagcgacat gagctcccga gagctgaaga ggcgctttga agtgtttggt gagattgagg 2820 agtgcgaggt gctgacaaga aataggagag gcgagaagta cggcttcatc acctaccggt 2880 gttctgagca cgcggccctc tctttgacaa agggcgctgc cctgaggaag cgcaacgagc 2940 cctccttcca gctgagctac ggagggctcc ggcacttctg ctggcccaga tacactgact 3000 acgattccaa ttcagaagag gcccttcctg cgtcagggaa aagcaagtat gaagccatgg 3060 attttgacag cttactgaaa gaggcccagc agagcctgca ttgataacag ccttaaccct 3120 cgaggaatac ctcaatacct cagacaaggc ccttccaata tgtttacgtt ttcaaagaaa 3180 tcaagtatat gaggagagcg agcgagcgtg agagaacacc cgtgagagag acttgaaact 3240 gctgtccttt aaaaaaaaaa aaaaaaa 3267 8 1023 PRT Homo sapiens 8 Met Ala Gly Asn Asp Cys Gly Ala Leu Leu Asp Glu Glu Leu Ser Ser 1 5 10 15 Phe Phe Leu Asn Tyr Leu Ala Asp Thr Gln Gly Gly Gly Ser Gly Glu 20 25 30 Glu Gln Leu Tyr Ala Asp Phe Pro Glu Leu Asp Leu Ser Gln Leu Asp 35 40 45 Ala Ser Asp Phe Asp Ser Ala Thr Cys Phe Gly Glu Leu Gln Trp Cys 50 55 60 Pro Glu Asn Ser Glu Thr Glu Pro Asn Gln Tyr Ser Pro Asp Asp Ser 65 70 75 80 Glu Leu Phe Gln Ile Asp Ser Glu Asn Glu Ala Leu Leu Ala Glu Leu 85 90 95 Thr Lys Thr Leu Asp Asp Ile Pro Glu Asp Asp Val Gly Leu Ala Ala 100 105 110 Phe Pro Ala Leu Asp Gly Gly Asp Ala Leu Ser Cys Thr Ser Ala Ser 115 120 125 Pro Ala Pro Ser Ser Ala Pro Pro Ser Pro Ala Pro Glu Lys Pro Ser 130 135 140 Ala Pro Ala Pro Glu Val Asp Glu Leu Ser Leu Leu Gln Lys Leu Leu 145 150 155 160 Leu Ala Thr Ser Tyr Pro Thr Ser Ser Ser Asp Thr Gln Lys Glu Gly 165 170 175 Thr Ala Trp Arg Gln Ala Gly Leu Arg Ser Lys Ser Gln Arg Pro Cys 180 185 190 Val Lys Ala Asp Ser Thr Gln Asp Lys Lys Ala Pro Met Met Gln Ser 195 200 205 Gln Ser Arg Ser Cys Thr Glu Leu His Lys His Leu Thr Ser Ala Gln 210 215 220 Cys Cys Leu Gln Asp Arg Gly Leu Gln Pro Pro Cys Leu Gln Ser Pro 225 230 235 240 Arg Leu Pro Ala Lys Glu Asp Lys Glu Pro Gly Glu Asp Cys Pro Ser 245 250 255 Pro Gln Pro Ala Pro Ala Ser Pro Gln Asp Ser Leu Ala Leu Gly Arg 260 265 270 Ala Asp Pro Gly Ala Pro Val Ser Gln Glu Asp Met Gln Ala Met Val 275 280 285 Gln Leu Ile Arg Tyr Met His Thr Tyr Cys Leu Pro Gln Arg Lys Leu 290 295 300 Pro Pro Gln Thr Pro Glu Pro Leu Pro Lys Ala Cys Ser Asn Pro Ser 305 310 315 320 Gln Gln Val Arg Ser Arg Pro Trp Ser Arg His His Ser Lys Ala Ser 325 330 335 Trp Ala Glu Phe Ser Ile Leu Arg Glu Leu Leu Ala Gln Asp Val Leu 340 345 350 Cys Asp Val Ser Lys Pro Tyr Arg Leu Ala Thr Pro Val Tyr Ala Ser 355 360 365 Leu Thr Pro Arg Ser Arg Pro Arg Pro Pro Lys Asp Ser Gln Ala Ser 370 375 380 Pro Gly Arg Pro Ser Ser Val Glu Glu Val Arg Ile Ala Ala Ser Pro 385 390 395 400 Lys Ser Thr Gly Pro Arg Pro Ser Leu Arg Pro Leu Arg Leu Glu Val 405 410 415 Lys Arg Glu Val Arg Arg Pro Ala Arg Leu Gln Gln Gln Glu Glu Glu 420 425 430 Asp Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Lys Glu Glu Glu 435 440 445 Glu Glu Trp Gly Arg Lys Arg Pro Gly Arg Gly Leu Pro Trp Thr Lys 450 455 460 Leu Gly Arg Lys Leu Glu Ser Ser Val Cys Pro Val Arg Arg Ser Arg 465 470 475 480 Arg Leu Asn Pro Glu Leu Gly Pro Trp Leu Thr Phe Ala Asp Glu Pro 485 490 495 Leu Val Pro Ser Glu Pro Gln Gly Ala Leu Pro Ser Leu Cys Leu Ala 500 505 510 Pro Lys Ala Tyr Asp Val Glu Arg Glu Leu Gly Ser Pro Thr Asp Glu 515 520 525 Asp Ser Gly Gln Asp Gln Gln Leu Leu Arg Gly Pro Gln Ile Pro Ala 530 535 540 Leu Glu Ser Pro Cys Glu Ser Gly Cys Gly Asp Met Asp Glu Asp Pro 545 550 555 560 Ser Cys Pro Gln Leu Pro Pro Arg Asp Ser Pro Arg Cys Leu Met Leu 565 570 575 Ala Leu Ser Gln Ser Asp Pro Thr Phe Gly Lys Lys Ser Phe Glu Gln 580 585 590 Thr Leu Thr Val Glu Leu Cys Gly Thr Ala Gly Leu Thr Pro Pro Thr 595 600 605 Thr Pro Pro Tyr Lys Pro Thr Glu Glu Asp Pro Phe Lys Pro Asp Ile 610 615 620 Lys His Ser Leu Gly Lys Glu Ile Ala Leu Ser Leu Pro Ser Pro Glu 625 630 635 640 Gly Leu Ser Leu Lys Ala Thr Pro Gly Ala Ala His Lys Leu Pro Lys 645 650 655 Lys His Pro Glu Arg Ser Glu Leu Leu Ser His Leu Arg His Ala Thr 660 665 670 Ala Gln Pro Ala Ser Gln Ala Gly Gln Lys Arg Pro Phe Ser Cys Ser 675 680 685 Phe Gly Asp His Asp Tyr Cys Gln Val Leu Arg Pro Glu Gly Val Leu 690 695 700 Gln Arg Lys Val Leu Arg Ser Trp Glu Pro Ser Gly Val His Leu Glu 705 710 715 720 Asp Trp Pro Gln Gln Gly Ala Pro Trp Ala Glu Ala Gln Ala Pro Gly 725 730 735 Arg Glu Glu Asp Arg Ser Cys Asp Ala Gly Ala Pro Pro Lys Asp Ser 740 745 750 Thr Leu Leu Arg Asp His Glu Ile Arg Ala Ser Leu Thr Lys His Phe 755 760 765 Gly Leu Leu Glu Thr Ala Leu Glu Glu Glu Asp Leu Ala Ser Cys Lys 770 775 780 Ser Pro Glu Tyr Asp Thr Val Phe Glu Asp Ser Ser Ser Ser Ser Gly 785 790 795 800 Glu Ser Ser Phe Leu Pro Glu Glu Glu Glu Glu Glu Gly Glu Glu Glu 805 810 815 Glu Glu Asp Asp Glu Glu Glu Asp Ser Gly Val Ser Pro Thr Cys Ser 820 825 830 Asp His Cys Pro Tyr Gln Ser Pro Pro Ser Lys Ala Asn Arg Gln Leu 835 840 845 Cys Ser Arg Ser Arg Ser Ser Ser Gly Ser Ser Pro Cys His Ser Trp 850 855 860 Ser Pro Ala Thr Arg Arg Asn Phe Arg Cys Glu Ser Arg Gly Pro Cys 865 870 875 880 Ser Asp Arg Thr Pro Ser Ile Arg His Ala Arg Lys Arg Arg Glu Lys 885 890 895 Ala Ile Gly Glu Gly Arg Val Val Tyr Ile Gln Asn Leu Ser Ser Asp 900 905 910 Met Ser Ser Arg Glu Leu Lys Arg Arg Phe Glu Val Phe Gly Glu Ile 915 920 925 Glu Glu Cys Glu Val Leu Thr Arg Asn Arg Arg Gly Glu Lys Tyr Gly 930 935 940 Phe Ile Thr Tyr Arg Cys Ser Glu His Ala Ala Leu Ser Leu Thr Lys 945 950 955 960 Gly Ala Ala Leu Arg Lys Arg Asn Glu Pro Ser Phe Gln Leu Ser Tyr 965 970 975 Gly Gly Leu Arg His Phe Cys Trp Pro Arg Tyr Thr Asp Tyr Asp Ser 980 985 990 Asn Ser Glu Glu Ala Leu Pro Ala Ser Gly Lys Ser Lys Tyr Glu Ala 995 1000 1005 Met Asp Phe Asp Ser Leu Leu Lys Glu Ala Gln Gln Ser Leu His 1010 1015 1020 9 3163 DNA Rattus norvegicus 9 atgcaggggg aagggaaggg tggggagtct ggagaggaac agttatgtgc tgacttgcca 60 gagctcgacc tctcccagct ggatgccagt gacttcgact cagccacgtg ctttggggag 120 ctgcagtggt gcccggagac ctcagagaca gagcccagcc agtacagccc tgatgattcc 180 gagttcttcc agattgacag tgagaatgaa gctctcttgg ctgcgcttac caagaccctg 240 gatgacatcc ccgaagacga tgtggggctg gctgccttcc caggactgga tgaaggcgac 300 acaccctcct gcaccccagc ttcacctgct cctttatctg tgccccccag ccccgccttg 360 gagaggcttc tgtccccagt gtctgaagtg gatgagcttt cactgctgca gaagctcctc 420 ctggccacat cctccccaac agcaagctct gatgctctga aggacggggc cacctggtcg 480 cagaccagcc tcagttccag aagtcagcgg ccttgtgtca aggtggatgg cacccaggac 540 aagaagaccc ccatgctacg gtctcagagc cggccttgta cagaactgca taagcacctc 600 acttcggtgc tgccctgccc caggggaaaa gcctgttccc cacctcccca cccaagtcct 660 cagctcctct ccaaagagga tgaggaggtg ggagaggatt gcccaagccc ctggccagct 720 ccagcgtctc cccaagactc actaggacag gacacggcca accccaacag tgcccaagtt 780 cccaaggacg acgtgagggc catggtacag ctcattcgct acatgcatac ctactgcctg 840 cctcagagga agctgcccca acgggcctca gagccaatcc cccagtcctg cagcagcccc 900 ttgaggaagg tcccaccccg atcccggcaa acccccaaag ccttctggac tgagttctcc 960 atcctaaggg aacttctggc ccaagatatc ctctgtgatg ttagcaagcc ctaccgcctg 1020 gccacacctg tctatgcttc tctcacaccc cagtccagaa ccaggccccc caaagacagt 1080 caggcctccc ctgcccactc tgccatggca gaagaggtga gaatcactgc ttcccccaag 1140 agcactggac ctagacccag cctccgtcct ctgaggctag aggtgaaacg ggatgtcaac 1200 aagcctgcaa ggcaaaagcg ggaggaagat gaggaggagg aagaggaaga agaagaggaa 1260 gaagaaaaag aggatgaaga agaggagtgg ggcaggaaga gaccaggtcg tggcctgcca 1320 tggaccaaac tagggaggaa gatggacagc tctgtgtgcc ctgtgcggcg ctccaggaga 1380 ctgaatccag agctgggccc ttggctgaca ttcactgatg agcccctagg tgctctaccc 1440 tcgatgtgcc tggctacaga gacccacgac ctggaagaag agctgggcgg cctcacagac 1500 agtagtcaag gccagcagct ccccctggga tcccagatcc ccaccctgga aagcccctgt 1560 gaaagtgggt gtggggacac agatgaagat ccaagctgcc cgcggccccc ttccagagac 1620 tcccccaggt gcctcatgct ggccttgtca caaagtgacc ctcttggcaa gaagagcttt 1680 gaggagtcct tgacagtgga gctttgtggc acagcaggac tcactccacc caccacacct 1740 ccatataagc ccatggagga ggaccccttc aagcaggaca ccaagcacag cccaggccaa 1800 gacacagctc ccagcctccc ttcccctgag actcttcagc tcacagccac cccaggggct 1860 tcccacaagc tgcccaagag gcacccggag cgaagtgagc tcctgtctca tctgcaacat 1920 gccacaaccc agccagtctc acaggctggc cagaagcgtc ccttctcctg ctcctttgga 1980 gaccatgact actgccaggt gatcaggcca gaggctgccc tgcagaggaa ggtgctgcgg 2040 tcctgggagc caatcaaggt ccaccttgaa gacttggccc accagggtgc aaccctgcca 2100 gtggaaacaa agacccctag gagggaggca gaccagaact gtgaccccac ccccaaggac 2160 agcatgcagc taagagacca tgagatccgt gccagcctca caaagcactt tgggctgctg 2220 gaaaccgctc tggaggagga agacttggct tcatgtaaaa gcccggagta tgacaccgta 2280 tttgaggaca gcagcagcag cagtggcgag agcagcttcc tgctagagga ggaggaagag 2340 gagggagggg aagaggacga tgaaggagag gactcagggg tcagccctcc ctgctccgac 2400 cactgcccct accagagccc acccagtaag gccagtcggc agctctgttc ccgaagccgc 2460 tccagttctg gctcctcatc ctgtagctcc tggtcaccag ctacccggaa gaacttcaga 2520 cttgagagca gagggccctg ttcagatgga accccaagcg cccggcatgc caagaagcgg 2580 cgggaaaagg ccatcggtga aggtcgtgtg gtatacatcc gaaatctctc cggtgacatg 2640 agctctcgag aactaaagaa gcgcttcgag gtgtttggtg agatagtcga gtgccaggtg 2700 ctgaggagaa gtaagagagg ccagaagcac ggttttatta ccttccggtg ttcggagcat 2760 gccgccctgt ccgtgaggaa cggcgctacc ctgagaaaac gcaatgagcc ctccttccac 2820 ctgagctatg gagggctccg gcacttccgc tggcccagat acaccgacta tgatcccacg 2880 tctgaagagt cccttccctc gtctgggaaa agcaagtacg aagccatgga ttttgacagc 2940 ttactgaaag aggcccagca gagcctgcat taatatcagc cttaaccttc gaggaatacc 3000 tcaatacctc agacaaggcc cttccaatat gtttacgttt tcaaagaaat gagtatatga 3060 ggaggagagc aagccaatga gcgagcgagc gagcgagcgt gagagaacac acaggagaga 3120 gagacttgaa tctgctgtcg tttcctttaa aaaaaaaaaa aaa 3163 10 990 PRT Rattus norvegicus 10 Met Gln Gly Glu Gly Lys Gly Gly Glu Ser Gly Glu Glu Gln Leu Cys 1 5 10 15 Ala Asp Leu Pro Glu Leu Asp Leu Ser Gln Leu Asp Ala Ser Asp Phe 20 25 30 Asp Ser Ala Thr Cys Phe Gly Glu Leu Gln Trp Cys Pro Glu Thr Ser 35 40 45 Glu Thr Glu Pro Ser Gln Tyr Ser Pro Asp Asp Ser Glu Phe Phe Gln 50 55 60 Ile Asp Ser Glu Asn Glu Ala Leu Leu Ala Ala Leu Thr Lys Thr Leu 65 70 75 80 Asp Asp Ile Pro Glu Asp Asp Val Gly Leu Ala Ala Phe Pro Gly Leu 85 90 95 Asp Glu Gly Asp Thr Pro Ser Cys Thr Pro Ala Ser Pro Ala Pro Leu 100 105 110 Ser Val Pro Pro Ser Pro Ala Leu Glu Arg Leu Leu Ser Pro Val Ser 115 120 125 Glu Val Asp Glu Leu Ser Leu Leu Gln Lys Leu Leu Leu Ala Thr Ser 130 135 140 Ser Pro Thr Ala Ser Ser Asp Ala Leu Lys Asp Gly Ala Thr Trp Ser 145 150 155 160 Gln Thr Ser Leu Ser Ser Arg Ser Gln Arg Pro Cys Val Lys Val Asp 165 170 175 Gly Thr Gln Asp Lys Lys Thr Pro Met Leu Arg Ser Gln Ser Arg Pro 180 185 190 Cys Thr Glu Leu His Lys His Leu Thr Ser Val Leu Pro Cys Pro Arg 195 200 205 Gly Lys Ala Cys Ser Pro Pro Pro His Pro Ser Pro Gln Leu Leu Ser 210 215 220 Lys Glu Asp Glu Glu Val Gly Glu Asp Cys Pro Ser Pro Trp Pro Ala 225 230 235 240 Pro Ala Ser Pro Gln Asp Ser Leu Gly Gln Asp Thr Ala Asn Pro Asn 245 250 255 Ser Ala Gln Val Pro Lys Asp Asp Val Arg Ala Met Val Gln Leu Ile 260 265 270 Arg Tyr Met His Thr Tyr Cys Leu Pro Gln Arg Lys Leu Pro Gln Arg 275 280 285 Ala Ser Glu Pro Ile Pro Gln Ser Cys Ser Ser Pro Leu Arg Lys Val 290 295 300 Pro Pro Arg Ser Arg Gln Thr Pro Lys Ala Phe Trp Thr Glu Phe Ser 305 310 315 320 Ile Leu Arg Glu Leu Leu Ala Gln Asp Ile Leu Cys Asp Val Ser Lys 325 330 335 Pro Tyr Arg Leu Ala Thr Pro Val Tyr Ala Ser Leu Thr Pro Gln Ser 340 345 350 Arg Thr Arg Pro Pro Lys Asp Ser Gln Ala Ser Pro Ala His Ser Ala 355 360 365 Met Ala Glu Glu Val Arg Ile Thr Ala Ser Pro Lys Ser Thr Gly Pro 370 375 380 Arg Pro Ser Leu Arg Pro Leu Arg Leu Glu Val Lys Arg Asp Val Asn 385 390 395 400 Lys Pro Ala Arg Gln Lys Arg Glu Glu Asp Glu Glu Glu Glu Glu Glu 405 410 415 Glu Glu Glu Glu Glu Glu Lys Glu Asp Glu Glu Glu Glu Trp Gly Arg 420 425 430 Lys Arg Pro Gly Arg Gly Leu Pro Trp Thr Lys Leu Gly Arg Lys Met 435 440 445 Asp Ser Ser Val Cys Pro Val Arg Arg Ser Arg Arg Leu Asn Pro Glu 450 455 460 Leu Gly Pro Trp Leu Thr Phe Thr Asp Glu Pro Leu Gly Ala Leu Pro 465 470 475 480 Ser Met Cys Leu Ala Thr Glu Thr His Asp Leu Glu Glu Glu Leu Gly 485 490 495 Gly Leu Thr Asp Ser Ser Gln Gly Gln Gln Leu Pro Leu Gly Ser Gln 500 505 510 Ile Pro Thr Leu Glu Ser Pro Cys Glu Ser Gly Cys Gly Asp Thr Asp 515 520 525 Glu Asp Pro Ser Cys Pro Arg Pro Pro Ser Arg Asp Ser Pro Arg Cys 530 535 540 Leu Met Leu Ala Leu Ser Gln Ser Asp Pro Leu Gly Lys Lys Ser Phe 545 550 555 560 Glu Glu Ser Leu Thr Val Glu Leu Cys Gly Thr Ala Gly Leu Thr Pro 565 570 575 Pro Thr Thr Pro Pro Tyr Lys Pro Met Glu Glu Asp Pro Phe Lys Gln 580 585 590 Asp Thr Lys His Ser Pro Gly Gln Asp Thr Ala Pro Ser Leu Pro Ser 595 600 605 Pro Glu Thr Leu Gln Leu Thr Ala Thr Pro Gly Ala Ser His Lys Leu 610 615 620 Pro Lys Arg His Pro Glu Arg Ser Glu Leu Leu Ser His Leu Gln His 625 630 635 640 Ala Thr Thr Gln Pro Val Ser Gln Ala Gly Gln Lys Arg Pro Phe Ser 645 650 655 Cys Ser Phe Gly Asp His Asp Tyr Cys Gln Val Ile Arg Pro Glu Ala 660 665 670 Ala Leu Gln Arg Lys Val Leu Arg Ser Trp Glu Pro Ile Lys Val His 675 680 685 Leu Glu Asp Leu Ala His Gln Gly Ala Thr Leu Pro Val Glu Thr Lys 690 695 700 Thr Pro Arg Arg Glu Ala Asp Gln Asn Cys Asp Pro Thr Pro Lys Asp 705 710 715 720 Ser Met Gln Leu Arg Asp His Glu Ile Arg Ala Ser Leu Thr Lys His 725 730 735 Phe Gly Leu Leu Glu Thr Ala Leu Glu Glu Glu Asp Leu Ala Ser Cys 740 745 750 Lys Ser Pro Glu Tyr Asp Thr Val Phe Glu Asp Ser Ser Ser Ser Ser 755 760 765 Gly Glu Ser Ser Phe Leu Leu Glu Glu Glu Glu Glu Glu Gly Gly Glu 770 775 780 Glu Asp Asp Glu Gly Glu Asp Ser Gly Val Ser Pro Pro Cys Ser Asp 785 790 795 800 His Cys Pro Tyr Gln Ser Pro Pro Ser Lys Ala Ser Arg Gln Leu Cys 805 810 815 Ser Arg Ser Arg Ser Ser Ser Gly Ser Ser Ser Cys Ser Ser Trp Ser 820 825 830 Pro Ala Thr Arg Lys Asn Phe Arg Leu Glu Ser Arg Gly Pro Cys Ser 835 840 845 Asp Gly Thr Pro Ser Ala Arg His Ala Lys Lys Arg Arg Glu Lys Ala 850 855 860 Ile Gly Glu Gly Arg Val Val Tyr Ile Arg Asn Leu Ser Gly Asp Met 865 870 875 880 Ser Ser Arg Glu Leu Lys Lys Arg Phe Glu Val Phe Gly Glu Ile Val 885 890 895 Glu Cys Gln Val Leu Arg Arg Ser Lys Arg Gly Gln Lys His Gly Phe 900 905 910 Ile Thr Phe Arg Cys Ser Glu His Ala Ala Leu Ser Val Arg Asn Gly 915 920 925 Ala Thr Leu Arg Lys Arg Asn Glu Pro Ser Phe His Leu Ser Tyr Gly 930 935 940 Gly Leu Arg His Phe Arg Trp Pro Arg Tyr Thr Asp Tyr Asp Pro Thr 945 950 955 960 Ser Glu Glu Ser Leu Pro Ser Ser Gly Lys Ser Lys Tyr Glu Ala Met 965 970 975 Asp Phe Asp Ser Leu Leu Lys Glu Ala Gln Gln Ser Leu His 980 985 990 11 20 DNA Artificial Sequence forward primer CME 9748 11 gtcacaaagc gacccaactt 20 12 22 DNA Artificial Sequence reverse primer CME 9749 12 gagtcatggt ctccaaagga ac 22 13 27 DNA Artificial Sequence AP1 adaptor primer 13 ccatcctaat acgactcact atagggc 27 14 22 DNA Artificial Sequence CME 9830 forward primer 14 gccactcgaa ggaacttcag at 22 15 22 DNA Artificial Sequence CME 9850 reverse primer B 15 gggttaaggc tgttatcaat gc 22 16 22 DNA Artificial Sequence CME 9831 reverse primer A 16 aggccagaag agaaacagga tg 22 17 22 DNA Artificial Sequence CME 9726 sequencing primer 17 cttctcctgt tcctttggag ac 22 18 20 DNA Artificial Sequence CME 9727 sequencing primer 18 tggggttcac ttgaggattg 20 19 22 DNA Artificial Sequence CME 9778 sequencing primer 19 attcaaaatc tcttccagcg ac 22 20 22 DNA Artificial Sequence CME 9776 sequencing primer 20 gaagacagaa gctgtgatgc tg 22 21 21 DNA Artificial SEquence SP1A primer 21 catcacagag cacgtcttga g 21 22 25 DNA Artificial Sequence SP2A primer 22 catgtagcgt atgagttgca ccatc 25 23 39 DNA Artificial Sequence Oligo d(T)-anchor primer 23 gaccacgcgt atcgatgtcg actttttttt ttttttttv 39 24 22 DNA Artificial Sequence PCR anchor primer 24 gaccacgcgt atcgatgtcg ac 22 25 29 DNA Artificial Sequence CVGI169 primer 25 ttgggtaacg ccagggtttt cccagtcac 29 26 29 DNA Artificial Sequence CVGI170 primer 26 ccccaggctt tacactttat gcttccggc 29 27 20 DNA Artificial Sequence CVGI171 primer 27 gccagtacag ccctgatgat 20 28 22 DNA Artificial Sequence CVGI172 primer 28 tccccagtgt ctgaagtgga tg 22 29 22 DNA Artificial Sequence CVGI281 primer 29 ctcattcgct acatgcatac ct 22 30 22 DNA Artificial Sequence CVGI282 primer 30 cggccttgtg tcaaggtgga tg 22 31 22 DNA Artificial Sequence CVGI283 primer 31 cttctggact gagttctcca tc 22 32 22 DNA Artificial Sequence CVGI390 primer 32 caggagactg aatccagagc tg 22 33 22 DNA Artificial Sequence CVGI391 primer 33 gacagtagtc aaggccagca gc 22 34 22 DNA Artificial Sequence CVGI457 primer 34 gagaccatga ctactgccag gt 22 35 22 DNA Artificial Sequence CVGI458 primer 35 accgctctgg aggaggaaga ct 22 36 22 DNA Artificial Sequence CVGI535 primer 36 ttaagcctta accctttgag ga 22 37 22 DNA Artificial Sequence CVGI536 37 ggcccagata caccgactat ga 22 38 20 DNA Artificial Sequence PGC-3 forward primer 38 tgctggccca gatacactga 20 39 21 DNA Artificial Sequence PGC-3 reverse primer 39 ggctgttatc aatgcaggct c 21 40 28 DNA Artificial Sequence PGC-3 probe 40 cgtcagggaa aagcaagtat gaagccat 28

Claims

1. An isolated and purified polynucleotide comprising a nucleic acid sequence which encodes a polypeptide having at least about 90% homology to a member selected from any one of

or

2. A polynucleotide which comprises the human PGC-3a cDNA sequence set out in SEQ ID NO:1 or a fragment thereof consisting of at least 8 bases.

3. A polynuclotide which comprises the human PGC-3b cDNA sequence set out in SEQ ID:NO:3 or a fragment thereof consisting of at least 8 bases.

4. A polynucleotide which comprises the human PGC-3c cDNA sequence set out in SED ID NO:7 or a fragment thereof consisting of at least 8 bases.

5. A homologue or orthologue of a polynucleotide according to any preceeding claim and having greater than 80% sequence homology to the to the PGC-3a cDNA sequence as set out in SEQ ID NO:1.

6. A homologue or orthologue of a polynucleotide according to any preceeding claim and having greater than 80% sequence homology to the to the PGC-3b cDNA sequence as set out in SEQ ID NO:3.

7. A homologue or orthologue of a polynucleotide according to any preceeding claim and having greater than 80% sequence homology to the to the PGC-3c cDNA sequence as set out in SEQ ID NO:7.

8. An isolated and purified polynucleotide molecule comprising a nucleic acid sequence which encodes a polypeptide having at least about 90% homology to any one of SEQ ID NO:10 positions 1-600, SEQ ID NO:10 positions 400-990, and SEQ ID NO:10 positions 200-800.

9. An expression vector comprising a polynucleotide according to any preceeding claim.

10. A transformed host cell comprising a polynucleotide according to any preceeding claim.

11. A purified polypeptide comprising the human PGC-3a amino acid sequence set out in SEQ ID NO.2 or a variant of SEQ ID NO.2 having at least about 90% homology to a member selected from (SEQ ID NO.2 positions 1-600, SEQ ID NO.2 positions 400-1002, SEQ ID NO.2 positions 200-800), or a biologically active fragment thereof.

12. A purified polypeptide comprising the human PGC-3b amino acid sequence set out in SEQ ID NO.4 or a variant of SEQ ID NO.4 having at least about 90% homology to a member selected from (SEQ ID NO.4 positions 1-600, SEQ ID NO.4 positions 400-996, SEQ ID NO.4 positions 200-800), or a biologically active fragment thereof.

13. A purified polypeptide comprising the human PGC-3c amino acid sequence set out in SEQ ID NO.8 or a variant of SEQ ID NO.8 having at least about 90% homology to a member selected from (SEQ ID NO.8 positions 1-600, SEQ ID NO.8 positions 400-1023, SEQ ID NO.8 positions 200-800), or a biologically active fragment thereof.

14. A purified polypeptide comprising the rat PGC-3 amino acid sequence set out in SEQ ID NO.10 or a variant of SEQ ID NO.10 having at least about 90% homology to a member selected from (SEQ ID NO.10 positions 1-600, SEQ ID NO.8 positions 400-990, SEQ ID NO.10 positions 200-800), or a biologically active fragment thereof.

15. A dominant negative mutant of a polypeptide according to any one of claims 11-14

16. A dominant positive mutants of a polypeptide according to any one of claims 11-14.

17. Antibodies specific for a polypeptide according to any one of claims 11-15.

18. A method for identifying a therapeutic agent capable of modulating the activity of PGC-3 for use in the regulation of metabolism, which method comprises:

or

and

19. A method as claimed in claim 18 and wherein the candidate compound modulator is contacted with a host-cell which expresses an PGC-3 polypeptide.

20. A PGC-3 modulator identified according to a method as claimed in claim 18 or claim 19.

21. A pharmaceutical composition which comprises a PGC-3 modulator as claimed in claim 20, or a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable diluent or carrier.

22. A method of treating a metabolic disease or medical condition mediated alone or in part by PGC-3, which comprises administering to a warm-blooded animal requiring such treatment an effective amount of an PGC-3 modulator as claimed in claim 20 or claim 21.

23. The use of an PGC-3 modulator as claimed in claim 20 in the production of a medicament for use in the treatment of a metabolic disease.